KR20170068458A - Treatment regimens using anti-nkg2a antibodies - Google Patents

Abstract

The present invention relates to a method of treating diseases, particularly cancer, using an antibody that specifically binds to and inhibits human NKG2A. Lt; RTI ID = 0.0 > anti-NKG2A < / RTI > antibodies.

Description

{TREATMENT REGIMENS USING ANTI-NKG2A ANTIBODIES}

Cross-references to related applications

This application claims the benefit of U.S. Provisional Application No. 62 / 050,948, filed September 16, 2014; 62 / 067,642, filed October 23, 2014; 62 / 083,929, filed November 25, 2014; 62 / 093,141, filed December 17, 2014; And 62 / 093,124, filed December 17, 2014, both of which are incorporated herein by reference in their entirety, including any drawings.

Reference to Sequence Listing

This application is filed with the sequence listing of the electronic form. The sequence list is 26 KB in size and is provided as a file with the title "NKG2A-T_ST25" generated on September 15, 2015. The information in the sequence listing of the electronic format is incorporated herein by reference in its entirety.

Technical field

The invention relates to the use of an anti-NSK2A antibody for therapy, in particular for the treatment of cancer.

NK cell activity is regulated by a complex mechanism involving both activation and suppression signals. Several distinct NK-specific receptors have been identified that play an important role in the recognition of NK cell mediated and the death of HLA class I deficient target cells. Natural Cytotoxic Receptor (NCR) refers to a family of activated receptor proteins and their expressing genes that are specifically expressed in NK cells. Examples of NCR include NKp30, NKp44, and NKp46 (see, for example, Lanier (2001) Nat Immunol 2: 23-27, the entire disclosure of which is incorporated herein by reference), Pende et al. 181: 787-796], Sivori et al (1997) J. Exp. Med. 186: 1129-1136], [ (Mandelboim et al. (2001) Nature 409: 1055-1060), which is incorporated herein by reference in its entirety). These receptors are members of the Ig phase and their cross-linking induced by specific mAbs leads to potent NK cell activation, resulting in increased intracellular Ca ⁺⁺ levels, triggering of cytotoxicity and lymphokine release and many types Lt; RTI ID = 0.0 > NK < / RTI > cytotoxicity to target cells.

CD94 / NKG2A is an inhibitory receptor observed in a subset of natural killer cells (NK cells), natural killer T cells (NKT cells) and T cells (α / β and γ / δ). CD94 / NKG2A limits cytokine release and cytotoxic response of the above-mentioned lymphocytes towards cells expressing CD94 / NKG2A-ligand HLA-E (see, for example, WO99 / 28748). In addition, HLA-E has been implicated in the development of specific tumor cells (Derre et al., J Immunol 2006; 177: 3100-7) and activated endothelial cells (Coupel et al., Blood 2007; 109: 2806-14) Lt; RTI ID = 0.0 > soluble form. ≪ / RTI > Antibodies that inhibit CD94 / NKG2A signaling may increase the cytokine release and cytolytic activity of lymphocytes towards HLA-E positive target cells, for example, the response of CD94 / NKG2A-positive NK cells towards viral infected cells have. Thus, therapeutic antibodies that inhibit CD94 / NKG2A but do not cause the death of CD94 / NKG2A-expressing cells (i.e., non-depleted antibodies) can induce modulation of tumor-growth in cancer patients. Furthermore, anti-NKG2A antibodies have also been proposed for use in the treatment of autoimmune or inflammatory diseases (see, e.g., US20030095965, WO2006070286).

Various antibodies to NKG2A are described in the art. In WO2006070286 and US 8,206,709 (see also WO2008 / 009545), the anti-NKG2A antibody Z270 has been described and in WO2009 / 092805 the humanized anti-NKG2A antibody Z199 has been described. Vance et al. (J Exp Med 1999; 190: 1801-12) was used in combination with rat anti-쥣 and NKG2-antibody 20D5 (currently available from BD Biosciences Pharmingen, catalog number 550518) ; U.S. Patent Application Publication 20030095965 discloses antibodies and antibody 3S9. Antibody Z270 does not neutralize the activation receptors NKG2C and NKG2E, but binds to the inhibitory receptor NKG2A and neutralizes it. Antibodies Z199, 20D5 and 3S9 all bind to activated NKG2 plus members NKG2C and NKG2E, in addition to NKG2A. Antibody Z270 blocks the binding of HLA-E to NKG2A while antibody Z199 neutralizes NKG2A without interfering with the binding of NKG2A to HLA-E.

We have found that the capacity and concentration of anti-NKG2A antibodies that completely occupy the NKG2A receptor on lymphocytes do not provide the maximum neutralization of the NKG2A receptor in vivo in the presence of HLA-E expressing target cells. In particular, the concentration of anti-NKG2A antibody required for complete neutralization of the NKG2A receptor on NK cells in the presence of HLA-E expressing target cells is greater than the concentration observed to completely occupy the NKG2A receptor in binding assays using NKG2A + Times higher. At the same time, it is observed that the anti-tumor effect of anti-NKG2A is increased as a function of the higher expression of HLA-E on tumor cells.

As shown herein, NKG2A + NK and CD8 + T lymphocytes are present in the tumor environment as well as in the circulatory system, and NKG2A + CD8 T cells are also significantly more potent in tumor-invading subsets than in CD8 + T cells in tumor draining lymph nodes and spleen And can be observed with increased frequency. Thus, the therapeutic regimens of the present invention further provide an advantageous method for treating solid tumors by modulating lymphocytes in the tumor environment using anti-NSG2A antibodies.

In one embodiment, there is provided a method of treating or preventing a disease (e.g. cancer, solid tumors, hematologic tumors) in an individual, the method comprising administering to the patient an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide, Wherein the anti-NKG2A antibody is administered to a subject having an anti-NKG2A antibody that is at least an EC ₅₀ for an NKG2A + NK cell response, optionally an anti-NKG2A antibody corresponding to EC ₁₀₀ (serum ) ≪ / RTI > concentration. In one embodiment, the amount is effective to achieve a concentration in an extravascular tissue (e.g., tumor tissue) of an anti-NKG2A antibody corresponding to at least an EC ₅₀ , and optionally an EC ₁₀₀ , for an NKG2A + NK cell response. In one embodiment, the NKG2A + NK cell response is assessed using assays of cytotoxic activity of NKG2A-expressing NK cells directed against HLA-E expressing target cells.

In one embodiment, there is provided a method of treating or preventing a disease (e.g. cancer, solid tumors, hematologic tumors) in an individual, the method comprising administering to the patient an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide, (Or alternatively at least 20, 30, 40, 50, 80 or 100 占 퐂 / ml) of an anti-NSG2A antibody, Of the anti-NKG2A antibody of the invention (and / or to maintain a steady state between two consecutive doses for a specific period of time and / or for a period of time).

Thus, in one embodiment, there is provided a method of treating or preventing a disease (e.g., cancer, solid tumors, hematological tumors) in an individual, the method comprising administering to NKG2A for at least one administration cycle, The method comprising administering to the subject an antibody that neutralizes the inhibitory activity of the polypeptide, wherein the cycle of administration comprises administering at least first and second (and optionally third, fourth, fifth, sixth, (E. G., The first and second, and / or eighth and eighth or more) administrations of the anti-NKG2A antibody to achieve a blood level of the anti-NSG2A antibody and / (E. G., When assessed by titrating an anti-NKG2A antibody on NKG2A-expressing cells in PBMC), the concentration of NKG2A < + > RTI ID = 0.0 > NKG2A < / RTI & 10 to 20 times, 10 to 50 times, 10 to 100 times, 20 to 50 times, or at least 10 times (for example, 10 to 20 times, 20 to 100 times, 30 to 100 times, 50 to 100 times), alternatively at least 50 times, 60 times, 80 times or 100 times. In one embodiment, the anti-NKG2A antibody competes with HLA-E for binding to human NKG2A.

In one embodiment, there is provided a method of treating or preventing disease (e.g., cancer, solid tumors, hematologic tumors) in an individual, the method comprising administering to a subject an effective amount of a compound that neutralizes the inhibitory activity of a human NKG2A polypeptide Comprising administering an antibody to a subject having a disease (e. G., Cancer, solid tumor), wherein the cycle of administration comprises administering at least first and second (and optionally third, fourth, (Or alternatively at least 20, 30, 40, or 50 μg / ml) of anti-NSG2A antibody, Is administered in an amount effective to achieve a blood (serum) concentration of the antibody or to maintain it between two consecutive administrations. In one embodiment, a particular continuous blood concentration is maintained, wherein the blood concentration does not fall below a substantially certain blood level for a certain period of time (e.g., between administrations of two antibodies), in other words, Although the concentration can vary over a certain period of time, the specific blood concentration that is maintained represents a minimum or "trough" concentration. In one embodiment, the therapeutically effective amount of an anti-NKG2A antibody is at least (about) at least about 1 week, at least about 2 weeks, or about 1 month after administration of the antibody. ₅₀ < / RTI > concentration, and optionally an EC ₁₀₀ concentration. In one embodiment, the anti-NKG2A antibody is administered at a dose of at least about 10 μg / ml (alternatively at least 20, 30, 40 or 50 μg / ml) for a period of at least about 1 week, (Serum) concentration, which enables significant "de-saturation" between two consecutive doses (successive doses can be separated, for example, one month, over two months). In one embodiment, wherein -NKG2A therapeutically active amount of the antibody in the tissue with respect to the following administration of the antibody at least one week or at least about 2 weeks NKG2A + NK cell response EC ₅₀ concentrations (at least), optionally substantially EC ₁₀₀ , Which in turn enables significant "de-saturation" between two successive administrations; Alternatively, the antibody is administered at about once every two months or at about once every two months. The serum concentration of the anti-NKG2A antibody during the de-saturation period is less than the specific concentration that should be achieved during the initial period (e. G., One week, two weeks, etc.); For example, the blood and / or tissue concentration of the anti-NKG2A antibody during the de-saturation period may be specified to be less than EC ₁₀₀ , and optionally less than EC ₅₀ for NKG2A + NK cell response.

In particular, in one embodiment where the NKG2A + T or NK cells in the extravascular tissue are to be regulated, for example, for the treatment of solid tumors, the anti-NKG2A antibody may be administered at the time of administration (e.g., ), Or an effective amount to achieve a peak concentration of at least about 40, 50, 60, 70 or 80 占 퐂 / ml, alternatively at least about 100 占 퐂 / ml, or to maintain its blood concentration. In one embodiment, the NKG2A antibody is administered in an effective amount to maintain a specific blood level for at least 1 week, and optionally at least 2 weeks after administration of the anti-NSG2A antibody. In particular, in one embodiment in which NKG2A + T or NK cells in an extravascular tissue are to be regulated for the treatment of, for example, solid tumors, the anti-NSK2A antibody is administered between the first and second (and optionally further) Or at least about 50, 60, 70 or 80 占 퐂 / ml, alternatively at least about 100 占 퐂 / ml, of the anti-NSK2A antibody. In one embodiment, consecutive doses (e. G., The first and second doses) are separated for at least 1 week, optionally about 2 weeks. The anti-NKG2A antibody may optionally be administered in an effective amount depending on the frequency of achieving or maintaining a blood concentration as specified for the entire duration of the dosing cycle.

In one embodiment, there is provided a method of treating or preventing a solid tumor in an individual, the method comprising administering to an individual having a solid tumor an antibody that neutralizes the inhibitory activity of the human NKG2A polypeptide for at least one administration cycle Wherein the cycle of administration comprises at least two administrations of an anti-NKG2A antibody, wherein the anti-NKG2A antibody comprises at least 4 [mu] g / ml, alternatively at least 10 [mu] g / (Minimal) concentration in the tumor environment). Alternatively, the anti-NKG2A antibody may be administered to achieve or maintain a (minimal) concentration within the extravascular tissue (e.g., within the tumor environment) of at least 4 [mu] g / ml, alternatively at least 10 [ ≪ / RTI > In one embodiment, the anti-NKG2A antibody is administered in an amount effective to maintain a sustained blood level of the anti-NSG2A antibody between two consecutive doses or at least 40 [mu] g / ml, alternatively at least 100 [ . In one embodiment, the anti-NKG2A antibody is administered at a dose of at least 40 μg / ml, optionally at least 100 μg / ml of the anti-NSK2A antibody (eg, at least one week, at least two weeks upon administration) , Followed by a period of time which allows for significant "de-saturation" of NKG2A-expressing cells in circulation during two consecutive doses, wherein the serum concentration of the anti- At least 40 [mu] g / ml, or at least 100 [mu] g / ml during the saturation period).

In one embodiment, there is provided a method of treating or preventing a hematologic tumor in a subject, the method comprising administering to a subject having a hematologic tumor an antibody that neutralizes the inhibitory activity of the human NKG2A polypeptide for at least one administration cycle Wherein the cycle of administration comprises at least two administrations of an anti-NKG2A antibody and wherein the anti-NSG2A antibody is administered at least 10 μg / ml of the continuous (minimal) blood concentration of the anti-NSG2A antibody between two consecutive doses Or < / RTI > Alternatively, the anti-NKG2A antibody is administered in an amount effective to achieve or maintain a sustained (minimal) blood level of at least 10 [mu] g / ml of anti-NSG2A antibody for the entire duration of the administration cycle.

In one embodiment, the antibody achieves a peak blood concentration of at least about 40 μg / ml, optionally about or at least about 100 μg / ml, at the time of administration (eg, on the day of administration, 24 hours or 48 hours of administration) Lt; / RTI >

In one embodiment, 40 ㎍ / ㎖ blood concentrations may provide the EC ₅₀ concentration for NKG2A + NK-cell responses in other vascular tissue. In one embodiment, a plasma concentration of 100 [mu] g / ml can provide an EC ₁₀₀ concentration for the NKG2A + NK cell reaction in tissue (outside of the vessel, e.g., within the tumor environment).

In one embodiment, the method of treatment comprises the steps of: (a) administering a first dose of an anti-NSG2A antibody between the first administration and subsequent administration of an anti-NSG2A antibody of at least about 40 [mu] g / ml, alternatively at least about 100 [ (Or loading) period to achieve or maintain a sustained (minimum) blood concentration, (b) a second lower dose of anti-NSG2A antibody is administered until the administration of the next anti-NSG2A antibody Treatment cycles using a maintenance period that is administered in an amount sufficient to achieve or maintain a sustained (minimal) blood level of the anti-NSG2A antibody of at least about 40 μg / ml, alternatively at least about 100 μg / . After the induction period, the maintenance period is followed. In one embodiment, the maintenance period comprises at least two administrations of the anti-NKG2A antibody. In one embodiment, the maintenance period is at least twice the dose and frequency of providing a continuous blood level of the anti-NSG2A antibody of at least about 40 [mu] g / ml, alternatively at least about 100 [mu] g / .

In certain embodiments, the blood concentration can be specified to be a serum concentration.

In one embodiment, the anti-NKG2A antibody substantially inhibits the inhibitory activity of human CD94 / NKG2A on NKG2A-positive lymphocytes in circulating or extravascular tissue in a human patient (in vivo) for about 1 week or about 2 weeks Fully neutralize.

In one embodiment, a method of treating an individual having cancer (e. G., A solid tumor) is provided, the method comprising administering to the subject an antibody that binds to NKG2A and neutralizes the inhibitory activity of NKG2A for at least one administration cycle Wherein the anti-NKG2A antibody is administered at a dose of 4 to 10 mg / kg, alternatively 4 to 6 mg / kg, alternatively 4 to 8 mg / kg, alternatively about 4 mg / kg, alternatively about 6 Kg / day, optionally in a dose of about 8 mg / kg, or alternatively about 10 mg / kg (body weight).

In one embodiment, a method of treating an individual having cancer (e. G., A solid tumor) is provided, the method comprising administering to the subject an antibody that binds to NKG2A and neutralizes the inhibitory activity of NKG2A for at least one administration cycle Wherein the method comprises the steps < RTI ID = 0.0 >

a. A loading period in which the antibody is administered intravenously at least once with an initial dose of 8 to 10 mg / kg, optionally about 10 mg / kg, and

b. Optionally, about 2 mg / kg, alternatively about 3 mg / kg, optionally about 2 mg / kg, alternatively about 2 mg / kg, alternatively from about 2 mg / Wherein the first administration within the maintenance period comprises two weeks after the initial dose and the second administration is within the maintenance period of at least two doses every two weeks, optionally at a dose of about 4 mg / kg or alternatively about 6 mg / kg (body weight) .

In one embodiment, the therapeutic regimen described herein is administered to an individual having a cancer, prior to the subject having undergone surgery to remove the cancer cells, i.e., the anti-NSG2A antibody therapy is used as a pre-operative therapy.

In one embodiment, the course of therapy or therapy designed to achieve a concentration of anti-NKG2A antibody corresponding to at least an EC ₅₀ , optionally EC ₁₀₀ , for an NKG2A + NK cell response in an extracellular tumor environment, Prior to surgery, in other words, it is administered to an individual having cancer as a pre-operative treatment.

In one embodiment, anti-NSK2A antibodies are administered to an individual having cancer cells expressing HLA-E on their surface. Alternatively, the HLA-E status of the cancer can be assessed prior to treatment with the anti-NSG2A antibody. In one embodiment, a combined method of HLA-E detection steps is provided to identify patients with HLA-E + tumors; These patients can then be treated with anti-NSG2A antibodies according to the treatment methods described herein.

In one embodiment of either the therapeutic use herein or the cancer treatment or prevention method, the treatment or prevention of cancer in an individual

a) determining the HLA-E polypeptide status of malignant cells in a subject having cancer, and

b) determining that the patient has an HLA-E polypeptide that is predominantly expressed on the surface of malignant cells, for example by binding to NKG2A and neutralizing the inhibitory activity of NKG2A according to any of the methods of treatment described herein RTI ID = 0.0 > anti-NKG2A < / RTI > Alternatively, the antibody inhibits binding of NKG2A by HLA-E.

In one embodiment of either the therapeutic use herein or the cancer treatment or prevention method, the treatment or prevention of cancer in an individual

a) determining the level of expression of an HLA-E nucleic acid or polypeptide of malignant cells in a subject having cancer, and

b) determining that the malignant cell selectively expresses an HLA-E nucleic acid or polypeptide at an increased level (e.g., high value, strong surface staining, etc.) relative to a reference level, for example, Comprising administering to the subject an anti-NKG2A antibody that binds to NKG2A and neutralizes the inhibitory activity of NKG2A, according to any one of < RTI ID = 0.0 > Alternatively, the antibody inhibits binding of NKG2A by HLA-E.

In one embodiment of any of the foregoing methods, determining the HLA-E polypeptide status or determining the expression level in step (a) comprises determining the level of expression of the HLA-E nucleic acid or polypeptide of the malignant cell in the biological sample, And comparing the level to a reference level (e. G., Value, weak cell surface staining, etc.). The reference level may be, for example, a healthy individual, a subject that does not elicit a clinical benefit from treatment with an anti-NKG2A antibody / does not induce a clinical benefit, or induces significant clinical benefit from treatment with an anti-NKG2A antibody It can correspond to an individual. The biological sample is tested for its ability to inhibit HLA-E nucleic acid or polypeptide at an increased level (e.g., a high value, a strong surface staining, a level corresponding to the level of an individual that derives significant clinical benefit from treatment with an anti-NSG2A antibody, A level that is higher than the level corresponding to an individual who does not elicit a clinical benefit / that induces a low clinical benefit from treatment with an NKG2A antibody, etc.) can be administered to a subject by, for example, RTI ID = 0.0 > anti-NKG2A < / RTI > antibody.

In one embodiment, the anti-NSG2A antibody is administered in a single agent therapy. In one embodiment, the anti-NKG2A antibody is administered in combination with one or more other anti-cancer agents.

In one embodiment of any of the therapeutic uses or therapeutic or prophylactic methods herein, the method further comprises administering to the subject a therapeutically active amount of a second anti-cancer agent. In one embodiment, the cancer is a solid tumor. In one embodiment, the second anticancer agent is an antibody that is capable of mediating ADCC (e. G., Binds to a human Fc receptor, e. G., CD16 via its Fc domain). In one embodiment, the antibody mediating ADCC is administered in an effective amount to induce (in vivo) antibody-dependent cellular cytotoxicity towards a human tumor cell in a human patient expressing a second anticancer drug-induced polypeptide.

In one embodiment, the second anticancer agent is an anti-EGFR antibody. In one embodiment, a patient treated with an anti-NKG2A antibody according to a Therapeutic regimen of the invention, in combination with an anti-EGFR antibody, has an insufficient response to a previous treatment with an anti-EGFR antibody (e. , Non-responders, or have a progressive disease) and have an undesirable prognosis for response to treatment with an anti-EGFR antibody (in the absence of treatment with anti-NSG2A).

In one embodiment, the combination is administered according to a particular clinical dosage regimen, particularly at a particular dosage level, and according to a particular dosage schedule (e.g., according to the prescribed dosage and / or the specific dosage schedule provided herein) Or for administration thereafter).

Antibodies (anti-NKG2A agonists) that neutralize the inhibitory activity of NKG2A polypeptides are antibodies that increase the ability of NKG2A-expressing NK and / or T cells to cause the death of HLA-E-expressing cells.

In one embodiment, the anti-NKG2A agonist reduces the inhibitory activity of NKG2A by blocking its ligand, HLA-E binding, in other words, the anti-NKG2A agonist interferes with the binding of NKG2A by HLA-E. Antibodies having the heavy chain of any of SEQ ID NOS: 2 to 6 and the light chain of SEQ ID NO: 7, respectively, are examples of such antibodies.

In one embodiment, the anti-NKG2A antibody is an antibody that binds NKG2A with significantly higher affinity for one or more activated NKG2 receptors. For example, in one embodiment, the antibody binds to NKG2A with significantly higher affinity for NKG2C. In a further or alternative embodiment, the antibody binds to NKG2A with significantly higher affinity for NKG2E. In a further or alternative embodiment, the antibody binds to NKG2A with significantly higher affinity for NKG2H. An antibody having the heavy chain of any one of SEQ ID NOS: 2 to 6 and the light chain of SEQ ID NO: 7 binds to NKG2A without substantial binding to NKG2C, NKG2E or NKG2H.

In a further or alternative embodiment, the anti-NKG2A antibody binds to the same epitope on NKG2A and / or binds to CD94 / NKG2A with the heavy chain of any of SEQ ID NOS: 2 to 6 and the light chain of SEQ ID NO: 7 ≪ / RTI > The antibody may be, for example, a human or humanized anti-NKG2A antibody.

In one embodiment, the anti-NKG2A antibody is a humanized antibody having a light chain CDR of any one of the heavy chain sequences of SEQ ID NO: 2 to 6 and a light chain CDR of the light chain sequence of SEQ ID NO: 7. An exemplary complementarity-determining region of such a humanized antibody having improved properties, such as lower immunogenicity, improved antigen-binding or other functional properties, and / or improved physicochemical properties such as better stability, (CDR) residues or sequences and / or sites for amino acid substitutions in the framework region (FR).

In other embodiments, pharmaceutical compositions and kits, and methods of use thereof, are provided.

These aspects are described more fully in the description of the invention provided herein, and additional aspects, features and advantages will be apparent from the description of the invention provided herein.

Figure 1 shows receptor occupancy in human blood of anti-NKG2A antibodies (humZ270, also referred to as IPH2201) at different concentrations (ng / ml) listed on the x-axis and MESF signals for receptor binding appear on the y- have. The antibody had a binding affinity (EC50) of about 4 ng / ml for NKG2A + cells (about 4 ng / ml of K _D ). This K _D is consistent with the K _D values observed in the other assays, particularly the affinity for binding to PBMC and affinity for recombinant NKG2A in the Biacore assay. The K _D (EC 100) for complete receptor occupancy was about 100 ng / ml.
Figure 2 shows the predicted receptor occupancy of anti-NSK2A antibodies based on human patients treated with a single dose of HumZ270 in the 92-patient Phase I study. Substantial (at least 90%) receptor saturation of NKG2A + cells can be achieved by administering 0.03 mg / kg every two weeks. Receptor saturation is greater with increasing dose and the plotted line in the figure with the lowest receptor saturation corresponds to the lowest dose (0.01 mg / kg); The higher dose level of each incremental formula then corresponds to the plotted line with the higher receptor saturation, and the line corresponding to the highest dose (10 mg / kg) has the highest receptor saturation. A dose of 0.1 mg / kg may be administered every four weeks to maintain saturation of substantially complete NKG2A.
Figure 3a shows the results from an autologous cell assay showing that the concentration of anti-NKG2A required for efficacy (blocking NKG2A in the presence of cells expressing its HLA-E ligand) is 100 times higher than the concentration that provides complete receptor saturation Show. In the CD107 mobilization assay, the EC50 concentration (the amount that provides 50% of the maximum) was determined to be about 400 ng / ml, and the EC100 was about 10,000 ng / ml (10 ug / ml).
Figure 3b shows the IPH2201 (anti-NSG2A) blood levels predicted after intravenous injection every two weeks, based on preliminary PK data and NCA analysis from the IPH2201 Phase I clinical trial. Different blood levels are achieved at different doses administered intravenously every two weeks. The IPH2201 concentration is greater as the dose is increased, and the plotted line in the plot with the lowest blood concentration corresponds to the lowest dose (0.01 mg / kg); The higher dose level of each incremental formula then corresponds to the plotted line providing the higher blood concentration, and the line corresponding to the highest dose (10 mg / kg) provides the highest blood concentration. A dose of 4 mg / kg is used for the efficacy of the tissue at an initial (peak) blood concentration of approximately 100 μg / ml, ie approximately EC ₁₀₀ and a sustained (minimum) blood concentration of greater than 30 μg / At the 4 dose point, a sustained (minimum) blood concentration of approximately 100 [mu] g / ml was provided. A dose of 10 mg / kg provided a sustained (minimum) blood concentration of approximately 100 [mu] g / ml.
Figure 3c shows the results of an intravenous injection every 2 weeks using a loading dose of 10 mg / kg (body weight) followed by a maintenance dose of 4 mg / kg (body weight), providing an initial and sustained blood concentration of approximately 100 [ The predicted IPH2201 blood concentration (indicated by the top line) is shown. For comparison, a fixed dose of 4 mg / kg is shown (bottom line in the figure) providing a continuous (or minimal) blood concentration of at least 30 [mu] g / ml at 2 weeks.
Figures 4A and 4B show the ability of anti-NSG2A to promote recognition of HNSCC cell line by NK cells. CD107 (upper) and CD137 (lower) FACS readings in the presence of the indicated target HNSCC cell line and on NKG2A-NK (left) or NKG2A + NK cells (right) in the presence or absence of anti-NSG2A at a concentration of 10 [mu] g / Value. Cell lines are sorted from left to right according to HLA-E surface expression levels. Each point represents PBMC from healthy volunteers. Figure 4a shows K562 targets with control and low or high levels of surface HLA-E, and Figure 4b demonstrates that anti-NSG2A can restore the dissolution of HNSCC with endogenous HLA-E expression. This effect is observed only in NKG2A-positive NK cells and depends on the level of HLA-E expression.
Figure 5 shows that the optimal dose of anti-NSG2A enhances ADCC by NK cells towards HNSCC FaDu cells induced by sub-optimal dose of anti-EGFR, cetuximab (ctx).
Figures 6A and 6B show the effect of increasing dose of anti-NSG2A and increasing dose of anti-EGFR (cetuximab). Figure 3A shows CD107 readings for control with and without target K562-HLA-E transfectant. Each healthy volunteer is represented by a different symbol: square or circle. The white symbols with crossed lines correspond to conditions in which the anti-NKG2A was replaced by a 10 [mu] g / ml hIgG4 isotype control co-incubated with 0.1 [mu] g / ml cetuximab. Figure 6b shows CD107 readings for HNSCC cell lines. For each concentration of cetuximab, the sign (square or circular) for the concentration of each anti-NSG2A was from left to right, 0 ug / ml, 0.1 ug / ml, 1 ug / ml and 10 ug / Corresponding.

Justice

The singular values used herein may mean one or more. The singular terms used in the claims (a) and (b) may mean one or more than one when used in conjunction with the term "comprises ". As used herein, "another" may mean at least a second or more.

When "comprising" is used, this may alternatively be replaced by " consisting essentially of "or" consisting of. &Quot;

NKG2A (OMIM 161555, the entire disclosure of which is incorporated herein by reference) is a member of the transcripts of the NKG2 group (Houchins, et al. (1991) J. Exp. Med. 173: 1017-1020). NKG2A is encoded by 7 exons over 25 kb, which shows some differential splicing. NKG2A, together with CD94, forms a heterodimeric suppressor receptor CD94 / NKG2A that is observed on the surface of NK cells, alpha / beta T cells, gamma / delta T cells and a subset of NKT cells. It has an ITIM within its cytoplasmic domain, similar to the inhibitory KIR receptor. As used herein, "NKG2A" refers to any mutant, derivative or small molecule of the NKG2A gene or encoded protein. In addition, any wild-type, full-length NKG2A that shares at least one biological characteristic or function and which shares at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide or amino acid identity Lt; / RTI > or protein sequence. Human NKG2A comprises 233 amino acids of the following sequence in three domains, the cytoplasmic domain comprising residues 1 to 70, the transmembrane domain comprising residues 71 to 93 and the extracellular domain comprising residues 94 to 233 Includes:

NKG2C (OMIM 602891, the entire disclosure of which is incorporated herein by reference) and NKG2E (OMIM 602892, the disclosure of which is incorporated herein by reference) are two other members of the transcripts of the NKG2 group (Gilenke, et al (1998) Immunogenetics 48: 163-173). The CD94 / NKG2C and CD94 / NKG2E receptors are activated receptors that are observed on the surface of a subset of lymphocytes such as NK cells and T-cells.

HLA-E (OMIM 143010, the entire disclosure of which is incorporated herein by reference) is expressed on the surface of a cell and is expressed as a ratio of the ratio of HLA- It is a typical MHC molecule. A soluble version of HLA-E was also identified. In addition to its T-cell receptor binding properties, HLA-E specifically binds to CD94 / NKG2A, CD94 / NKG2B, and CD94 / NKG2C, thereby enhancing the expression of natural killer (NK) (see, for example, Braud et al. (1998) Nature 391: 795-799, the entire disclosure of which is incorporated herein by reference). Surface expression of HLA-E prevents target cells from being lysed by CD94 / NKG2A + NK, T, or NKT cell clones. As used herein, "HLA-E" refers to any variant, derivative or small molecule of the HLA-E gene or encoded protein. Also, it is preferred that at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the nucleotide or amino acid identity ≪ / RTI >

In the context of the present invention, "CD94 / NKG2A positive lymphocyte" refers to cells of the lymphoid lineage (e.g., NK-, NKT- and T- cells) expressing CD94 / NKG2A on the cell surface, Lt; RTI ID = 0.0 > CD94 < / RTI > and NKG2A, or / and an antibody specifically recognizing an epitope on NKG2A alone. "CD94 / NKG2A-positive lymphocytes" also include immortalized cell lines of lymphatic origin (eg, NKL, NK-92).

In the context of the present invention, the intracellular pathway leading to a lymphocyte response, such as cytokine release and cytotoxic response, "reduces the inhibitory activity of NKG2A", "neutralizes NKG2A" or "neutralizes the inhibitory activity of NKG2A"Lt; RTI ID = 0.0 > CD94 / NKG2A < / RTI > This can be measured, for example, in NK- or T-cell based cytotoxicity assays wherein the ability of the therapeutic compound to stimulate the death of HLA-E positive cells by CD94 / NKG2A positive lymphocytes is measured. In one embodiment, the antibody agent is at least 10% increase in cytotoxicity of CD94 / NKG2A-restricted lymphocytes in association with the described cytotoxicity assay, preferably at least 40% or 50% increase in lymphocyte cytotoxicity, or more preferably, Resulting in at least a 70% increase in NK cytotoxicity. If the anti-NKG2A antibody reduces or blocks the interaction of CD94 / NKG2A and HLA-E, it can increase the cytotoxicity of CD94 / NKG2A-restricted lymphocytes. This can be assessed, for example, in a standard 4-hour in vitro cytotoxicity assay using NK cells expressing CD94 / NKG2A and target cells expressing HLA-E. Such NK cells do not efficiently kill targets that express HLA-E, since CD94 / NKG2A recognizes HLA-E and results in initiation and amplification of inhibitory signaling that prevents lymphocyte-mediated cell lysis. Such in vitro cytotoxicity assays are described, for example, in Coligan et al., Eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), which is well known in the art. Chromium release and / or other parameters for evaluating the ability of the lymphocyte to stimulate target cells, e. G., P815, K562 cells or appropriate tumor cells, to kill are also described in Sivori et al., J. Exp. Med. 1997; 186: 1129-1136; Vitale et al., J. Exp. Med. 1998; 187: 2065-2072; Pessino et al. J. Exp. Med. 1998; 188: 953-960); Neri et al. Clin. Diag. Lab. Immun. 2001; 8: 1131-1135); Pende et al. J. Exp. Med. 1999; 190: 1505-1516, the entire disclosure of each of which is incorporated herein by reference. Prior to the addition of NK cells, the target cells are labeled with ⁵¹ Cr and the killing is estimated as a consequence of the killing in proportion to the release of ⁵¹ Cr from the cells to the medium. The addition of antibodies that prevent CD94 / NKG2A from binding to HLA-E results in the initiation and inhibition of amplification of inhibitory signaling through CD94 / NKG2A. Thus, the addition of such agents results in an increase in the lymphocyte-mediated killing of the target cells. Thereby, at this stage, agents that prevent CD94 / NKG2A-induced negative signal transduction, for example by blocking ligand binding, are identified. In certain ⁵¹ Cr-releasing cytotoxicity assays, CD94 / NKG2A-expressing NK effector cells can kill HLA-E-negative LCL 721.221 target cells, but HLA-E expressing LCL 721.221-Cw3 control cells are significantly less Kill. In contrast, YTS effector cells lacking CD94 / NKG2A efficiently kill both cell lines. Thus, NK effector cells less efficiently kill HLA-E ⁺ LCL 721.221-Cw3 cells due to HLA-E-induced inhibitory signaling through CD94 / NKG2A. When the NK cells were preincubated with the blocking anti-CD94 / NKG2A antibody according to the invention in such a ⁵¹ Cr-releasing cytotoxicity assay, the HLA-E-expressing LCL 721.221-Cw3 cells were incubated in an antibody-concentration- More efficiently. In addition, the inhibitory activity (i.e., cytotoxic potency) of an anti-NKG2A antibody can be measured in any of a number of different ways, for example, in Sivori et al., J. Exp. Med. 1997; 186: 1129-1136. ≪ / RTI > Activation of NK cytotoxicity can be assessed, for example, by measuring the increase in cytokine production (e. G., IFN-y production) or cytotoxicity markers (e. G., CD107 or CD137 mobilization). In an exemplary protocol, IFN-y production from PBMC is assessed by cell surface and intracellular staining and analysis by flow cytometry after 4 days in culture. Briefly, Brefeldin A (Sigma Aldrich) is added at a final concentration of 5 [mu] g / ml during the last 4 hours of culture. CD3 and < RTI ID = 0.0 > CD3 < / RTI > prior to staining using cells for permeabilization (IntraPrep ™; Beckman Coulter) and PE-anti-IFN-y or PE-IgG1 (Pharmingen) Incubate with anti-CD56 mAb. Generation of GM-CSF and IFN-y from polyclonal activated NK cells was assessed by ELISA (GM-CSF: DuoSet Elisa, R & D Systems, Minneapolis, MN, IFN- Amp; nummin.) In a supernatant.

Within this entire specification, "treatment of cancer" and the like are always referred to with respect to the anti-NKG2A binding agent (e. G., An antibody) and, depending on the patent subject matter enabling the patent in the country ) (For at least one treatment) (preferably in a pharmaceutically acceptable carrier material) an anti-NKG2A binding agent to a subject in need of such treatment, a mammal, in particular a human, (Therapeutically effective amount), preferably in a dose (amount) as specified herein; (b) the use of an anti-NSG2A binding agent for the treatment of cancer (particularly in humans), or an anti-NSG2A binding agent for use in the treatment; (c) the use of an anti-NKG2A binding agent for the manufacture of a pharmaceutical formulation for the treatment of cancer, combining the anti-NKG2A binding agent with a pharmaceutically acceptable carrier, for the manufacture of a pharmaceutical formulation for the treatment of cancer A pharmaceutical formulation comprising an effective amount of an anti-NKG2A binding agent suitable for use of the anti-NKG2A binding agent or for the treatment of cancer; Or (d) any combination of a), b) and c).

The term "biopsy" as used herein is defined as the removal of tissue for the purpose of, for example, testing to establish a diagnosis. Examples of types of biopsies include, for example, application of suction through an injection needle attached to a syringe; Removing tissue fragments by instrument; Using an appropriate instrument to remove through an endoscope; For example, surgical resection of the entire lesion; And the like.

The term "antibody" as used herein refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain within the heavy chain, the antibody is assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these are further divided into subclasses such as IgG1, IgG2, IgG3, IgG4, etc., or isotype. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50 to 70 kDa). The N-terminus of each chain defines a variable region of about 100 to more than 110 amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to these light and heavy chains, respectively. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as "alpha", "delta", "epsilon", "gamma", and "mu", respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG is a class of exemplary antibodies used herein because it is the most common antibody in physiological situations and is most easily prepared in a laboratory setting. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human or otherwise human-compatible. "Antibody" also includes any fragment or derivative of any of the antibodies described herein.

The term "specifically binds to" means that when evaluating using recombinant forms of a protein, either an epitope within it, or a native protein present on the surface of an isolated target cell, the antibody is preferably conjugated ≪ / RTI > to a partner, e. G., NKG2A. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected by radioactive labeling, physical methods such as mass spectrometry or direct or indirect fluorescent labeling detected using, for example, cell fluorescence assay (e.g., FACScan). Binding in excess of the amount observed in the control, nonspecific agent indicates that the agent binds to the target. Agents that specifically bind to NKG2A can bind to either NKG2A alone or as a dimer with CD94.

When an antibody is said to "compete" with a particular monoclonal antibody, it is preferred that the antibody is monoclonal in a binding assay using either a recombinant molecule (e.g., NKG2A) or a surface expression molecule (e.g., NKG2A) It means to compete with null antibody. For example, if the test antibody reduces the binding of the heavy chain of any of SEQ ID NOS: 2 to 6 and the light chain of SEQ ID NO: 7 to the NKG2A polypeptide or NKG2A-expressing cell in the binding assay, Each is said to "compete" with such an antibody.

As used herein, the term "affinity" refers to the strength of binding of an antibody to an epitope. The affinity of the antibody is provided by a dissociation constant, Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody- And [Ag] is the molar concentration of the unbound antigen. The affinity constant K _a is defined by 1 / Kd. Methods for determining the affinity of mAbs have been described by Harigan, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds. Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993) and Muller, Meth. Enzymol. 92: 589-601 (1983), which references are incorporated herein by reference in their entirety. One standard method widely known in the art for determining the affinity of a mAb is to use surface plasmon resonance (SPR) screening (e.g., by analysis using a BIAcore (TM) SPR analyzer).

In the context of the present application, "crystal site" refers to the interaction or binding site on the polypeptide.

The term "epitope" refers to an antigenic determinant site and is a region or region on an antigen to which the antibody binds. Protein epitopes can include amino acid residues directly involved in binding and amino acid residues that are efficiently blocked by a particular antigen binding antibody or peptide, i. E., Amino acid residues in the "put-print" of the antibody. It is the simplest form or minimal structural region on a complex antigen molecule that can be combined with, for example, an antibody or a receptor. The epitope may be linear or steric / structural. The term "linear epitope" is defined as an epitope consisting of consecutive amino acid residues in the linear sequence (primary structure) of an amino acid. The term "steric or structural epitope" is defined as an epitope consisting of all non-contiguous amino acid residues and is thus a separate part of the linear sequence of amino acids that are adjacent to each other by folding of the molecule (secondary, tertiary and / Structure). The three-dimensional epitope depends on the three-dimensional structure. Thus, the term "steric" is often used interchangeably with "structural ".

The term "agonist" is used herein to refer to compounds, mixtures of compounds, biological macromolecules, or extracts made from biological materials. The term "therapeutic agent" refers to an agent having biological activity.

For purposes herein, a "humanized" or "human" antibody refers to an antibody in which the constant and variable framework regions of one or more human immunoglobulins are fused with the binding region of an animal immunoglobulin, eg, a CDR. Such an antibody is designed to maintain the binding specificity of the non-human antibody from which the binding domain is derived, but to avoid an immune response to the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered " to produce specific human antibodies in response to antigen challenge infections (see, e. G., Green et al. (1994) Nature Genet 7:13); See, for example, Taylor et al. (1994) Int Immun 6: 579, the entire teachings of which are incorporated herein by reference). Lonberg et al. (1994) Nature 368: 856; Complete human antibodies can also be constructed by gene or chromosomal transfection methods and phage display techniques, all of which are well known in the art (see, e.g., McCafferty et al. (1990) Nature 348: 552-553 ] Reference). Human antibodies may also be produced by in vitro activated B cells (see, for example, U.S. Patent Nos. 5,567,610 and 5,229,275, the contents of which are incorporated herein by reference).

A "chimeric antibody" refers to a chimeric antibody that (a) has a constant region or a portion thereof altered, substituted or exchanged so that the antigen binding region (variable region) has a different or altered class, a constant region of effector function and / Such as enzymes, toxins, hormones, growth factors, drugs and the like; Or (b) the variable region, or a portion thereof, is altered, substituted or exchanged into a variable region having a different or altered antigen specificity.

The term "Fc domain," " Fc portion ", and "Fc region" refer to the C- terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of a human gamma (E. G., Alpha, delta, epsilon and mu for human antibodies) or their naturally occurring alleles refer to their corresponding sequences in allotypes. Unless otherwise specified, Kabat amino acid numbering that is commonly accepted for immunoglobulins is used in the present disclosure (Kabat et al. (1991) Sequences of Protein of Im- munological Interest, 5th ed. , United States Public Health Service, National Institute of Health, Bethesda, MD).

The term "isolated "," purified ", or "biologically pure" refers to a substance that is substantially or essentially free of components that normally accompany it, as observed in its native state. Purity and homogeneity are typically determined using analytical chemical techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The predominant species present in the preparation is substantially purified.

The terms "polypeptide "," peptide ", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This term applies to amino acid polymers wherein one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, and to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "recombinant" when used in reference to a cell, or nucleic acid, protein or vector, means that the cell, nucleic acid, protein or vector is modified by introduction of a heterologous nucleic acid or protein, Lt; RTI ID = 0.0 > cells. ≪ / RTI > Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell, or otherwise expresses a native gene that is abnormally expressed, expressed or not expressed at all.

Within the context of this disclosure, the term " binding "antibody to a polypeptide or epitope refers to an antibody that has specificity and / or affinity and binds to the crystal site.

The term "identity" or "same" refers to the degree of sequence relatedness between polypeptides as determined by the number of matches between strings of two or more amino acid residues when used in the context of the sequences of two or more polypeptides. "Identity" is an identical percent match between the smaller of two or more sequences with a gap alignment (if present) handled by a particular mathematical model or computer program (i.e., "algorithm"). The identity of the related polypeptides can be readily calculated by known methods. Such methods are described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; And Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

The method for determining identity is designed to provide maximum agreement between the sequences tested. Methods for determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences are described in GAP (Devereux et al., Nucl. Acid Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, ), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from NCBI (National Center for Biotechnology Information) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; It is possible. The well-known Smith Waterman algorithm can also be used to determine identity.

Generation of antibodies

Anti-NKG2A agonists bind to the extracellular portion of the human CD94 / NKG2A receptor and reduce the inhibitory activity of human CD94 / NKG2A receptors expressed on the surface of CD94 / NKG2A positive lymphocytes. In one embodiment, the agonist competes with HLA-E for binding to CD94 / NKG2A, i.e., the agonist interferes with and reduces the interaction between CD94 / NKG2A and its ligand HLA-E. The antibody may bind to a combined epitope on CD94 and NKG2A or an epitope on NKG2A alone. In one embodiment, the antibody binds to an epitope on NKG2A that at least partially overlaps the HLA-E binding site.

In one embodiment, the anti-NKG2A agonist is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody. In one embodiment, the agonist comprises a constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 antibody. In one embodiment, the agent is a fragment of an antibody selected from IgA, IgD, IgG, IgE, and IgM antibodies. In one aspect, the agents are Fab fragments, Fab 'fragments, Fab'-SH fragments, F (ab) 2 fragments, F (ab') 2 fragment, Fv fragment, a heavy chain Ig (llama or camel Ig), V _HH fragments, Single domain FV and single chain antibody fragment. In one embodiment, the agonists are scFV, dsFV, minibodies, diabodies, triabodies, kaparabodies, IgNARs; And a multispecific antibody.

Preferably, the anti-NKG2A antibody does not show significant specific binding to an Fc [gamma] receptor, e.g., CD16. Such antibodies may comprise constant regions of various heavy chains that are not known to bind to Fc receptors. One such example is the human IgG4 constant region. In one embodiment, the IgG4 antibody comprises a modification to prevent the formation of a half antibody (fab arm exchange) in vivo, for example, the antibody has a position according to EU-index Including the mutation from serine to proline at residue 241 corresponding to SEQ ID NO: 228 (Kabat et al., "Sequences of proteins of immunological interest", 5th ed., NIH, Bethesda, ML, 1991 ]). Such modified IgG4 antibodies will remain intact in vivo and retain bivalent (high affinity) binding to NKG2A, as opposed to native IgG4, which undergoes in vivo fab arm exchange to bind NKG2A in a monovalent manner, The binding affinity can be changed. Alternatively, antibody fragments, e.g., Fab or F (ab ') 2 fragments, that do not comprise one or more constant regions can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing the binding of an antibody to an Fc receptor protein in a BIACORE assay. In addition, any human antibody type (e.g., IgG1, IgG2, IgG3, or IgG4) can be used, wherein the Fc portion is modified to minimize or eliminate binding to Fc receptors See WO03101485, which is incorporated herein by reference). Assays for assessing Fc receptor binding, for example, cell-based assays are well known in the art and are described, for example, in WO03101485.

An anti-NKG2A antibody can advantageously bind to the extracellular portion of NKG2A with a KD that is at least 100 fold lower than the KD for binding to NKG2C. In one embodiment, the antibody binds to the extracellular portion of NKG2A with a KD of at least 150, 200, 300, 400 or 10,000 times lower than the KD for binding to NKG2C. In another embodiment, the antibody binds to the extracellular portion of NKG2A with a KD at least 100 fold lower than the KD for binding to NKG2C, NKG2E and / or NKG2H molecules. In a further embodiment, the antibody binds to the extracellular portion of NKG2A with a KD of at least 150, 200, 300, 400 or 10,000 times lower than the KD for binding to NKG2C, NKG2C and / or NKG2H molecules. This can be measured, for example, in a Biacore experiment, where the ability of the agonist to bind to the extracellular portion of immobilized CD94 / NKG2A (e.g., purified from CD94 / NKG2 expressing cells or generated in the biosystem) Were measured and compared to binding of agonists to CD94 / NKG2C and / or other CD94 / NKG2 variants similarly produced in the same assay. Alternatively, CD94 / NKG2A may be naturally expressed (e.g., after transient or stable transfection), the binding of the antibody to cells overexpressing it measured, and the CD94 / NKG2C and / or other CD94 / NKG2 variants Lt; RTI ID = 0.0 > cell < / RTI > The anti-NKG2A antibody is optionally capable of binding to NKG2B, and NKG2B is an NKG2A splice variant that forms an inhibitory receptor with CD94. In one embodiment, the affinity can be determined, for example, by binding to an NKG2A-CD94-Fc fusion protein covalently immobilized by Biacore as shown in Example 8 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference. Can be measured using the method disclosed in U.S. Patent No. 8,206,709.

The antibody may be administered in a single dose, for example, in the range of 0.5 to 10 ng / ml, alternatively 1 to 5 ng / ml, alternatively 1 to 10 ng / ml, alternatively 1 to 20 ng / ml, for binding to the NKG2A- expressing cells (high affinity) can have EC _50. The NKG2A-expressing cells may be, for example, NKG2A-expressing cells in human PBMC. In one embodiment, the NKG2A-expressing cells are stably overexpressed cells prepared to express CD94 / NKG2A, such as CD94 / NKG2A, as shown in Example 13 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference. Gt; Ba / F3 < / RTI > In one embodiment, the antibody binding affinity (K _D) has an, optionally, less than 10 ^-9 M, alternatively, less than 10 ^-10 M, or optionally less than 10 ^-11 M, optionally, ^10-10 M to 10 ^-12 M, alternatively, a binding affinity of 10 ^-10 M to 10 ^-11 M is bivalent to the human NKG2A polypeptide. Affinity may be assessed for binding to short chain NKG2A-CD94-mFc constructs as described, for example, in U.S. Patent No. 7,932,055, the disclosure of which is incorporated herein by reference.

Anti-NKG2A antibodies include, for example, the VH human acceptor framework from human acceptor sequences selected from VH1_18, VH5_a, VH5_51, VH1_f and VH1_46, and the JH6 J-segment or other human germline VH framework Or a human or humanized antibody comprising a sequence. The VL region human acceptor sequence may be, for example, VKI_O2 / JK4.

In one embodiment, the antibody is a humanized antibody based on antibody Z270. The different humanized Z270 VH chains are shown in SEQ ID NOs: 2-6 (variable domain amino acids are underlined). The humanized Z270VH light chain is shown in SEQ ID NO: 7. HumZ270 antibodies are also disclosed in U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference. HumZ270VH6 (SEQ ID NO: 2) is based on VH5_51; HumZ270VH1 (SEQ ID NO: 3) is based on VH1_18; humZ270VH5 (SEQ ID NO: 4) is based on VH5_a; humZ270VH7 (SEQ ID NO: 5) is based on VH1_f; humZ270VH8 (SEQ ID NO: 6) is based on VH1_46; All have a JH6 J-segment. Each of these antibodies retains a high affinity binding to NKG2A, and because the six C-terminal amino acid residues of each Kabat CDR-H2 of the humanized construct are identical to the human acceptor framework, the host immune response to the antibody The possibility is low. Using the alignment program VectorNTI, the following sequence identity was obtained between humZ270VH1 and humZ270VH5, -6, -7 and -8: 78.2% (VH1 to VH5), 79.0% (VH1 to VH6), 88.7% VH7) and 96.0% (VH1 vs. VH8).

In one embodiment, the agent is selected from the group consisting of: (i) an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98% And (ii) a light chain variable region of an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: . In one embodiment, the agent is selected from the group consisting of: (i) an amino acid sequence of any one of SEQ ID NOS: 2 to 6, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98% And (ii) an amino acid sequence of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: Lt; / RTI > An antibody having a heavy chain comprising the sequence of any one of SEQ ID NOS: 2 to 6 and a light chain comprising the sequence of SEQ ID NO: 7 neutralizes the inhibitory activity of NKG2A, but substantially inhibits the activation of receptors NKG2C, NKGE or NKG2H Do not combine. These antibodies further compete with HLA-E for binding to NKG2A on the surface of the cells. In one embodiment, the agonist comprises an HCDR1, HCDR2 and / or HCDR3 sequence derived from a heavy chain having the amino acid sequence of any of SEQ ID NOS: 2-6. In one aspect of the invention, the agent comprises an LCDR1, LCDR2 and / or LCDR3 sequence derived from a light chain having the amino acid sequence of SEQ ID NO: 7.

Heavy chain (Underlined in the variable area)

In one embodiment, the anti-NKG2A antibody comprises a CDR-H1 corresponding to residues 31-35 (amino acid sequence SYWMN (SEQ ID NO: 8)) of any of SEQ ID NOs: 2-6, SEQ ID NOs: 2-6 (SEQ ID NO: 9)) (optionally, 50-66 if the six terminal amino acids of human origin are included, i.e. the sequence RIDPYDSETHYSPSFQG (SEQ ID NO: 9) in the VH6 heavy chain) (Amino acid sequence GGYDFDVGTLYWFFDV (SEQ ID NO: 10) corresponding to the amino acid sequence of SEQ ID NO: 2 to 6, CDR-H2 corresponding to the sequence RIDPYDSETHYAQKLQG NO: 12) (95 to 102 according to Kabat). In one embodiment, CDR-H2 corresponds to residues 50-66 of any of SEQ ID NOs: 2-6. Alternatively, the CDR may comprise 1, 2, 3, 4 or more amino acid substitutions.

In one embodiment, the anti-NKG2A antibody comprises CDR-L1 corresponding to residues 24-34 (amino acid sequence RASENIYSYLA (SEQ ID NO: 13)) of SEQ ID NO: 7, residues 50-56 L3, which corresponds to CDR-L2 corresponding to SEQ ID NO: 14 (SEQ ID NO: 14) and residues 89 to 97 (amino acid sequence QHHYGTPRT (SEQ ID NO: 15)) of SEQ ID NO: 7 . Alternatively, the CDR may comprise 1, 2, 3, 4 or more amino acid substitutions.

In one embodiment, the anti-NKG2A antibody comprises CDR-H1 corresponding to residues 31-35 of any of SEQ ID NOS: 2-6, residues 50-60 of any of SEQ ID NOS: 2-6 (alternatively, CDR-H3 corresponding to residues 99 to 114 (95 to 102 according to Kabat) of any of SEQ ID NOS: 2 to 6, a residue of SEQ ID NO: 7 An antibody comprising CDR-L3 corresponding to amino acid residues 24-34, CDR-L2 corresponding to residues 50-56 of SEQ ID NO: 7, and CDR-L3 corresponding to residues 89-97 of SEQ ID NO: 7.

In one embodiment, the agent is a fully human antibody directed against the CD94 / NKG2A epitope to which any of the above-mentioned antibodies bind.

Although the above-mentioned antibodies can be used, it will be appreciated that other antibodies can be produced. For example, NKG2A, preferably but not exclusively, any fragment of human NKG2A or any combination of NKG2A fragments can be used as an immunogen to induce antibodies, wherein the antibodies bind to NKG2A expressing NK cells Lt; RTI ID = 0.0 > NKG2A < / RTI > polypeptide. Most preferably, the epitope is an epitope specifically recognized by an antibody having the heavy chain of any of SEQ ID NOS: 2 to 6 and the light chain of SEQ ID NO: 7.

In one embodiment, the agonist competes with the humZ270 antibody disclosed in U.S. Patent No. 8,206,709 (the disclosure of which is incorporated herein by reference) in conjunction with the extracellular portion of the human CD94 / NKG2A receptor. Competitive binding can be measured, for example, in the BiaCore experiment, where the extracellular portion of the immobilized CD94 / NKG2A receptor (e. G. Purified from CD94 / NKG2 expressing cells or produced in the bio-system) saturated with humZ270 ≪ / RTI > is measured. Alternatively, expression of the CD94 / NKG2A receptor naturally, overexpressed (e. G., After transient or stable transfection), and binding of the agonist to cells pre-incubated with saturating capacity of Z270 is measured. In one embodiment, competitive binding is assessed by binding to Ba / F3-CD94-NKG2A cells by flow cytometry as shown for example in Example 15 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference. By using the method disclosed in U.S. Patent No. 8,206,709.

The anti-NKG2A antibody may be incorporated into the pharmaceutical formulation at a concentration of from 1 mg / ml to 500 mg / ml, wherein the formulation has a pH of from 2.0 to 10.0. The formulations may additionally comprise buffering agents, preservative (s), isotonic agent (s), chelating agent (s), stabilizers and surfactants. In one embodiment, the pharmaceutical formulation is a formulation comprising an aqueous formulation, i.e., water. Such formulations are typically solutions or suspensions. In a further embodiment, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50 w / w% water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 w / w% water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 w / w% water.

In another embodiment, the pharmaceutical formulation is a lyophilized formulation, wherein the solvent or diluent is added to the medical or patient prior to use.

In other embodiments, the pharmaceutical formulation is a dried formulation (e. G., Freeze-dried or spray-dried) that is ready for use without any prior dissolution.

In a further aspect, the pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present at a concentration of at least 1 mg / ml and the formulation has a pH of from about 2.0 to about 10.0.

In other embodiments, the pH of the formulation ranges from about 2.0 to about 10.0, from about 3.0 to about 9.0, from about 4.0 to about 8.5, from about 5.0 to about 8.0, and from about 5.5 to about 7.5.

In a further embodiment the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citric acid salt, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogenphosphate, sodium phosphate and tris (hydroxymethyl) Is selected from the group consisting of bicine, tricine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffer constitutes an alternative embodiment of the present invention.

In a further embodiment, the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment, the formulation further comprises an isotonic agent. In a further embodiment, the formulation also comprises a chelating agent. In a further embodiment of the invention, the formulation further comprises a stabilizer. In a further embodiment, the formulation further comprises a surfactant. For convenience, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

It is possible that other ingredients may be present in the pharmaceutical pharmaceutical formulations of the present invention. Such additional components include, but are not limited to, wetting agents, emulsifiers, antioxidants, bulking agents, wall thickening agents, chelating agents, metal ions, oily vehicles, proteins (e.g., human serum albumin, gelatin or protein) For example, amino acids such as betaine, taurine, arginine, glycine, lysine, and histidine. Of course, such additional ingredients should not adversely affect the overall stability of the pharmaceutical formulations of the present invention.

A pharmaceutical composition containing an antibody may be administered to a patient at some site, e.g., a local site, e.g., a skin and mucous membrane site, a bypass site to be absorbed, such as an artery, vein, For example, it may be administered to a patient in need of such treatment through skin, subcutaneous, muscular or abdominal administration. Administration of the pharmaceutical composition may be by any of several routes of administration such as subcutaneous, intramuscular, intraperitoneal, intravenous, tongue, sublingual, buccal, oral, oral, intrathecal, nasal, Can be made to patients in need of such treatment through the epidermis, dermis, percutaneous, vaginal, rectal, ocular, such as conjunctiva, urethra, and parenteral,

Diagnosis and treatment of malignant tumors

Methods useful for diagnosis, prognosis, monitoring, treatment and prevention of cancer in an individual are described. Although the methods described herein are particularly useful for the treatment of solid tumors, the therapeutic regimens described herein can also be used for a variety of hematologic cancers and infectious diseases and inflammation and autoimmune disorders. The methods and compositions of the present invention can be used to treat cancer, including, for example, carcinomas of the skin including bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and squamous cell carcinoma; Leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma and bucket lymphoma, and multiple myeloma Hematopoietic tumors of the lymphatic system; Hematopoietic tumors including acute and chronic myelogenous leukemia, promyelocytic leukemia, and myelodysplastic syndrome; Tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; Other tumors including melanoma, testicular, teratoma, neuroblastoma and glioma; Tumors of the central and peripheral nervous system including astrocytomas, neuroblastomas, gliomas and schwannomas; Tumors of mesenchymal origin, including fibrous sarcoma, rhabdomyosarcoma, and osteosarcoma; And various cancers and other proliferative diseases including, but not limited to, melanoma, pigmented skin dryness, keratocystosis, testicular tumors, thyroid follicular cancer and other tumors including malignant carcinomas.

Examples of lymphoid lineage hematopoietic tumors include, but are not limited to, T-cell disorders such as, for example, T-cell lymphoblastic leukemia (T-PLL) Cell and B-cell tumors; Preferably T-cell type large granular lymphocytic leukemia (LGL); Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); T-NHL liver spleen lymphoma; Peripheral / thymic T cell lymphoma (polymorphisms and subclass types of immunocompetent cells); Vascular < / RTI > immunoblot T-cell lymphoma; Vascular centric (nasal) T-cell lymphoma; Malignant (Ki 1+) giant cell lymphoma; Superficial T-cell lymphoma; T-lymphocyte; Lymphoma / leukemia (T-Lbly / T-ALL), and multiple myeloma.

In one embodiment, the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, the HNSCC is an oral pharyngeal tumor, a laryngeal tumor, a tumor of the oral cavity, or a tumor of the hypopharynx. In one embodiment, the HNSCC is oral SCC (OCSCC). OCSCC includes squamous cell carcinoma of the lips, anterior two-thirds of the tongue, floor of the mouth, ball mucosa, gums, palate, and molar back triangles. In one embodiment, the HNSCC is a metastatic cancer.

When treating an individual having a solid tumor, a compound (e. G., An antibody) that neutralizes the inhibitory activity of a human NKG2A polypeptide is advantageously administered to a subject having no or currently undergoing surgery to remove cancer cells, May be administered to a subject having cancer that is not undergoing such surgery for a period of time. However, it will be appreciated that the compound may also be administered to a patient who has undergone surgery to remove cancer cells, or whose operation is underway. When the anti-NKG2A compound is administered to an individual who has not undergone surgical intervention to remove cancer cells (e.g., to remove HNSCC cells), the NKG2A-binding compound may be administered to a subject, for example, 8 weeks. In one embodiment, at least one (e.g., one, two, three or more times) complete dosing cycle (s) of treatment with the anti-NSG2A compound is administered prior to surgery. In one embodiment, the administration cycle is from 2 weeks to 8 weeks.

In one embodiment, the cancer treated by the methods disclosed herein is an HLA-E-expressing cancer.

Patients with cancer can be treated with the anti-NSG2A agonist with or without previous detection steps to assess the expression of HLA-E on the surface of tumor cells. Advantageously, the method of treatment may comprise detecting HLA-E nucleic acids or polypeptides (e. G., On tumor cells) in a biological sample of the tumor from the subject. Examples of biological samples include any suitable biological fluid (e. G., Serum, lymph, blood), cell samples or tissue samples. For example, the tissue sample may be a tumor tissue or a sample of tumor adjacent tissue. Alternatively, the HLA-E polypeptide is detected on the surface of malignant cells. A biological sample expresses HLA-E (for example, expresses HLA-E significantly; high level of reference level, expresses HLA-E with high intensity staining using anti-HLA-E antibody) The determination indicates that the individual has cancer that can have a strong benefit from the treatment of an agent that inhibits NKG2A. In one embodiment, the method comprises the steps of determining the level of expression of an HLA-E nucleic acid or polypeptide in a biological sample and comparing the level to a reference level (e.g., a predetermined value, a weak cell surface stain, etc.) ). ≪ / RTI > The determination that a biological sample expresses an HLA-E nucleic acid or polypeptide at an increased level relative to a reference level indicates that the individual has a cancer that can be treated with an agent that inhibits NKG2A.

In one embodiment, the determination that a biological sample (e. G., A sample comprising a tumor cell, tumor tissue and / or tumor adjacent tissue) significantly expresses an HLA-E nucleic acid or polypeptide indicates that the individual is treated with an agent that inhibits NKG2A Quot ;, and " cancer " "Significantly expressed" when referring to an HLA-E polypeptide means that the HLA-E polypeptide is expressed in a significant number of tumor cells taken from a given individual. Although the definition of the term "significantly expressed" is not bound by an exact percentage value, in some instances, a receptor that is "markedly expressed" is at least 30%, 40%, 50%, 60% 70%, 80% or more.

Determining whether an individual has cancer cells expressing an HLA-E polypeptide can be accomplished, for example, by obtaining a biological sample (e.g., by performing a biopsy) from an individual comprising a cancer cell, -E < / RTI > polypeptide, and detecting whether the cells express HLA-E on their surface. Optionally, determining whether an individual has cancer cells expressing HLA-E comprises performing an immunohistochemistry assay. Optionally, determining whether an individual has cancer cells expressing HLA-E comprises conducting a flow cytometric assay.

In an exemplary embodiment, a mammalian host having a detectable level of cancer cells comprising administering an anti-NSG2A antibody at a dose and frequency in accordance with the disclosure sufficient to detectably reduce the progression of the cancer in the host For example, a method of reducing the progression of cancer in a human patient is provided.

In one embodiment, there is provided a method of treating or preventing a disease (e.g., hematologic tumor, inflammation or autoimmune disease, infection) in an individual, comprising administering an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide (E. G., 90%, 95%) receptor saturation in PBMC, e.g., by titrating the anti-NKG2A antibody on NKG2A-expressing cells, 20, 30, or 50 times higher circulating levels than the control. The antibody may be incubated with NKG2A-expressing cells in human PBMC for example, 0.5 to 10 ng / ml, alternatively 1 to 10 ng / ml, alternatively 1 to 20 ng / ml, for example, about 4 ng / for binding it may have an EC _50.

In one embodiment, there is provided a method of treating or preventing a disease (e.g., a solid tumor, an inflammatory or autoimmune disease, an invention) in an individual, the method comprising administering an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide (E. G., 90%, 95%) receptor saturation when assessed by titrating the anti-NKG2A antibody on NKG2A-expressing cells in PBMCs for at least 10, 20, 30 or 50 To an individual having the disease in an amount that achieves a concentration within the extravascular extravasary tissue (e. G., A tumor or tumor environment).

The EC ₅₀ for the NKG2A + NK cell response of the blocking anti-NKG2A antibody HumZ270 is about 4 ug / ml, so that the amount of anti-NKG2A antibody to achieve and / or maintain a blood level of at least 4 ug / . Advantageously, the subject is administered an anti-NSK2A antibody in an amount sufficient to achieve and / or maintain a blood level of at least 10 [mu] g / ml (EC ₁₀₀ for NKG2A + NK cell response) in the subject. For example, the blood concentration to be achieved and / or maintained may be in the range of 10 to 12 [mu] g / ml, 10 to 15 [mu] g / ml, 10 to 20 [ 10 to 100 占 퐂 / ml, 10 to 150 占 퐂 / ml, or 10 to 200 占 퐂 / ml. In one embodiment, a tissue concentration of at least about 4 μg / ml (EC ₅₀ for NKG2A + NK cell response) or optionally at least about 10 μg / ml (EC ₁₀₀ for NKG2A + NK cell response) is achieved in the subject and / or An amount of an anti-NKG2A antibody is administered to an individual. (For example, in the treatment of solid tumors) an amount of anti-NSG2A antibody is administered to achieve and / or maintain a tissue concentration of at least 10 [mu] g / ml when tissue outside the vasculature is targeted; For example, administering an anti-NSG2A antibody in an amount sufficient to achieve a blood level of at least 100 [mu] g / ml is expected to achieve an extravascular tissue (e.g., tumor tissue) concentration of at least 10 [mu] g / . For example, blood concentrations that must be achieved and / or maintained to achieve / maintain 10 [mu] g / ml in a tissue are 100-110 [mu] g / ml, 100-120 [ 100 to 150 占 퐂 / ml, 100 to 200 占 퐂 / ml, 100 to 250 占 퐂 / ml, or 100 to 300 占 퐂 / ml.

In some embodiments, at least an EC ₅₀ for an NKG2A + NK cell reaction, Optionally an anti-NSK2A antibody is administered to obtain a blood (serum) concentration corresponding to a drug or at least about EC ₁₀₀ . The NKG2A + NK cell response can be assessed using a suitable assay of cytotoxic activity of NKG2A-expressing NK cells directed against HLA-E expressing target cells. Examples include assays based on the markers of NK cell activation, such as CD107 or CD137 expression, as shown in the Examples herein. "EC ₅₀ " for NKG2A + NK cell response refers to the effective concentration of anti-NSG2A antibody producing 50% of its maximal response or effect on such NKG2A + NK cell response. "EC ₁₀₀ " for NKG2A + NK cell response refers to the effective concentration of anti-NKG2A antibody that produces its substantially maximal response or effect with respect to such NKG2A + NK cell response. For the treatment of in some implementations, in particular solid tumors, which is achieved the concentration is, for the outside, examples of the (blood vessel structure in the NKG2A + NK cells, which at least corresponds to the EC _50, optionally, from about or at least about EC ₁₀₀ for reaction, Tumor or < / RTI > tumor environment).

Suitable therapeutic protocols for treating humans include, for example, administering to a patient an effective amount of an anti-NSG2A antibody, wherein the method comprises at least one cycle of administration, wherein at least one dose of the anti-NSG2A antibody Optionally 2 to 10 mg / kg, alternatively 4 to 10 mg / kg, alternatively 6 to 10 mg / kg, alternatively 8 to 10 mg / kg, alternatively 2 to 4 mg / kg, alternatively 4 to 6 mg / Kg, alternatively from 4 to 8 mg / kg, alternatively from 6 to 8 mg / kg (body weight). Optionally, at least 2, 3, 4, 5, 6, 7 or 8 times the dose of anti-NSK2A antibody is administered. In one embodiment, the administration cycle is from 2 weeks to 8 weeks. In one embodiment, the dosing cycle is 8 weeks. In one embodiment, the cycle of administration is 8 weeks and comprises administering an anti-NSK2A antibody at a dose of once every two weeks (i.e., a total of four doses).

In one embodiment of any of the embodiments herein, the anti-NKG2A antibody is administered about once every two weeks.

Suitable therapeutic protocols for treating humans include, for example, administering to a patient an effective amount of an anti-NSG2A antibody, wherein the antibody is administered twice monthly and between at least two consecutive doses of the anti-NSG2A antibody An effective amount to maintain a sustained blood level of the anti-NSG2A antibody of at least 10 [mu] g / ml is 2 to 10 mg / kg, alternatively 2 to 6 mg / kg, alternatively 2 to 8 mg / Kg, alternatively 2 to 3 mg / kg, or alternatively about 2, 3 or 4 mg / kg (body weight). These doses may be optionally administered to provide a sustained blood level of at least 10 [mu] g / ml anti-NSG2A antibody throughout the treatment cycle. Achieving a blood concentration of 10 [mu] g / ml anti-NSG2A antibody corresponds to an EC ₁₀₀ for an antibody, e.g., humanized Z270.

Additional suitable treatment protocols for treating humans include, for example, administering to a patient an effective amount of an anti-NKG2A antibody, wherein the antibody is administered twice a month and comprises at least two consecutive doses of anti- An effective amount of 2 to 10 mg / kg, alternatively 2 to 8 mg / kg, alternatively 2 to 6 mg / kg, optionally, 2 to 10 mg / kg, to maintain a sustained serum concentration of the anti-NSG2A antibody between at least 40 & To 4 mg / kg, optionally 2 to 3 mg / kg or alternatively about 2, 3 or 4 mg / kg (body weight). These doses may be optionally administered to provide a sustained blood level of at least 40 [mu] g / ml anti-NSG2A antibody throughout the treatment cycle. Achieving a blood concentration of 40 [mu] g / ml of anti-NKG2A antibody can in turn result in an approximately 4 [mu] g / ml tissue corresponding to an EC ₅₀ for an antibody, e.g., NKG2A + NK cell response to humanized Z270 ≪ / RTI > extracellular tissue, tumorous environment of solid tumors).

Additional advantageous suitable treatment protocols for treating humans include, for example, administering to a patient an effective amount of an anti-NKG2A antibody, wherein the antibody is administered twice a month and comprises at least two consecutive An effective amount to maintain a sustained blood level of the anti-NSG2A antibody between at least 100 [mu] g / ml between administration is 4 to 10 mg / kg, alternatively 4 to 6 mg / kg, alternatively 4 to 8 mg / About 4 mg / kg, optionally about 6 mg / kg, alternatively about 8 mg / kg or alternatively about 10 mg / kg (body weight). These doses may optionally be administered throughout the treatment cycle to provide a sustained serum concentration of at least 100 [mu] g / ml anti-NSG2A antibody. To achieve a blood level of the antibody, wherein -NKG2A 100 ㎍ / ㎖ The antibodies in turn, for example, a tissue of about 10 ㎍ / ㎖ equivalent to EC ₁₀₀ for humanized Z270 (e. G., Vascular et al., Tumor Environment) ≪ / RTI > concentration.

A further advantageous suitable treatment protocol for treating a human with cancer includes a therapy using a maintenance period followed by a loading period using a larger dose. For example, the loading period may comprise administering to the patient an effective amount of an anti-NKG2A antibody, wherein the antibody is administered at least 100 μg / ml of the anti-NSK2A antibody Administered at least once in an effective amount to maintain blood concentration. For example, if administered in a single dose, an anti-NSK2A antibody with a loading dose of 10 mg / kg may be administered, and the first administration of an anti-NSK2A antibody in a maintenance regimen may take about 2 weeks (or less ). The maintenance regimen may then use lower doses and / or lower doses to maintain a sustained blood level of at least 100 [mu] g / ml of anti-NSG2A antibody between consecutive doses in maintenance therapy. For example, the maintenance therapy may be administered at a rate of 2 to 10 mg / kg, alternatively 4 to 10 mg / kg, alternatively 2 to 4 mg / kg, alternatively 4 to 6 mg / kg, alternatively 4 to 8 mg / Kg / kg, optionally about 4 mg / kg, alternatively about 6 mg / kg, alternatively about 8 mg / kg (body weight).

In one embodiment, the anti-NKG2A antibody is administered to the NKG2A + NK cell response for at least about 1 week, at least about 2 weeks, without significant "destruction" of NKG2A on the cycle during at least two consecutive administrations of anti- (Serum) and / or tissue (e.g., tumor tissue) corresponding to at least an EC ₅₀ , and optionally a drug, or at least about EC ₁₀₀ . In another aspect, the antibody for at least about one week, at least about 2 weeks NKG2A + NK, at least in EC _50, selective for cellular response of about or at least the blood corresponding to about EC ₁₀₀ (serum) and / or tissue while (for example, , Tumor tissue), which allows for a significant "de-saturation" of NKG2A on the cycle during two consecutive doses of the anti-NKG2A antibody (or between each successive dose) do. For example, an anti-NKG2A antibody can be administered in an amount and frequency that can result in at least 50% destruction of NKG2A on the cell during the course of the treatment, for example, between two consecutive administrations of anti-NKG2A antibody have. In one example, the amount that enables a meaningful "de-saturation" of NKG2A on the circulating cells is an amount that provides a blood concentration of less than EC ₅₀ for the NKG2A + NK cell response, optionally less than EC ₂₀ for the NKG2A + NK cell response.

An advantageous suitable therapeutic protocol for treating a human comprises, for example, administering to a patient an effective amount of an anti-NKG2A antibody, wherein the antibody is administered for at least about 1 week or at least about 2 weeks for at least 10 < RTI ID = / Ml or 100 [mu] g / ml of anti-NKG2A antibody in the presence of an effective amount of NKG2A in the circulation during two consecutive administrations of the anti-NKG2A antibody (or during each administration) Enabling significant "de-saturation" (e. G., Leading to this).

For example, the antibody may be administered at least once a month (or less than once every two months), and at least about 10 μg / ml, 40 μg / ml or 100 μg / ml for at least about 1 week or at least about 2 weeks An effective amount to achieve a blood level of the anti-NKG2A antibody is 2 to 10 mg / kg, alternatively 2 to 8 mg / kg, alternatively 2 to 6 mg / kg, alternatively 2 to 4 mg / To 3 mg / kg, or alternatively about 2, 3 or 4 mg / kg (body weight).

Saturation (and de-saturation) of NKG2A on the circulating cells can be evaluated by obtaining peripheral blood samples and using standard methods for assessing receptor occupancy.

In another embodiment, following the step of administering to the patient one or more first therapeutic agents (e.g., an inducer, such as a chemotherapeutic agent) in an amount and in an amount sufficient to effect the reaction (partial or complete response) There is provided a method of reducing the risk of cancer progression, comprising administering to the patient a predetermined amount of an anti-NSG2A antibody at a dose and frequency according to the disclosure, Or to provide a therapeutic regimen for the reduction of cancer progression in a human patient.

In a further embodiment, a composition comprising an anti-NKG2A antibody is administered to a subject at a dose and frequency according to the disclosure to promote cancer remission in the subject, for example cancer in a human patient A method of promoting remission of the subject is provided. In a further aspect, there is provided the use of a composition comprising an anti-NSG2A antibody at a dose and frequency according to the disclosure, to an individual, comprising the step of promoting cancer remission in the subject, There is provided a method of preventing recurrence of cancer in a remission state after avascular treatment.

In a further aspect, there is provided a method of reducing the risk of developing cancer, comprising administering a prophylactically effective amount of an anti-NKG2A antibody to a subject at a dose and frequency according to the disclosure to achieve the desired physiological effect (s) , A method of reducing the time to the onset of the cancer disease and / or a method of reducing the severity of the cancer diagnosed at an early stage.

In a further aspect, there is provided a method of increasing survivability over an appropriate period of time in a human patient diagnosed with cancer. In another aspect, there is provided a method for improving the quality of life of a cancer patient comprising administering to the patient an effective amount of a composition for improving the quality of life of the patient. In a further aspect, for example, the method described herein can be applied to reduce the total number of cancer cells, thereby significantly reducing the number of cancer cells in a vertebrate host. In a related sense, there is provided a method of killing (e.g., directly or indirectly inducing its death) cancer cells in a vertebrate animal, for example a human cancer patient.

The anti-NKG2A antibody may be administered in a monotherapy or in a secondary or concurrent administration (co-administration) with a second therapeutic agent, for example, an anti-EGFR antibody. Auxiliary or concurrent administration includes simultaneous administration of the compounds in the same or different dosage forms or separate administration (e. G., Sequential administration) of the compounds. Thus, the anti-NKG2A agonist and the second therapeutic agent can be co-administered in a single formulation. Alternatively, the anti-NSG2A agonist and the second therapeutic agent may be formulated for separate administration and administered simultaneously or sequentially. The second therapeutic agent will usually be administered in an amount and treatment regimen typically used for the agent in a monotherapy for the particular disease or disorder being treated.

Example

Example  1-humanized Z270 Biacore  analysis

Humanized Z270 (humZ270) is described in U.S. Patent No. 8,206,709 (Novo Nordisk), the disclosure of which is incorporated herein by reference. As described in U.S. Patent No. 8,206,709, the HumZ270 VKI_O2 / JK4 light chain and various heavy chain acceptor frameworks were generated as human IgG4 antibodies. The heavy chain framework includes "VH1" based on VH1_18 / JH6; "VH5" based on VH5_a; "VH6" based on VH5_51; "VH7" based on VH1_f; And "VH8" based on VH1_46, all having a JH6 J-segment. Antigen-binding properties were analyzed in Biacore T100 (Biacore AB, Uppsala, Sweden). Antigens in the form of short chain NKG2A-CD94-mFc constructs are reacted with an amine group using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide And covalently immobilized on a sensor CM5 chip (Biacore Abbe, Uppsala, Sweden). The immobilization level was targeted to 300 RU. Z270 antibody variants were incubated with a series of concentrations (0.157, 0.313, 0.625, 1.25 (0.157), 0.325 , 2.5 nM). All samples were then injected onto the immobilized antigen for 2 minutes at a flow rate of 40 l / min. Thereafter, running buffer was injected at 40 ㎕ / min for 3 minutes for antibody dissociation analysis. After each run, the remaining antibody from the antigen was completely removed by injection of a regeneration buffer (10 mM NaOH, 500 mM NaCl) (30 sec, 10 [mu] l / min). Data were evaluated using the Biacore T100 evaluation software.

The affinity of humanized Z270 VH1 was determined to be 67 pM. The affinities of VH5, VH6, VH7 and VH8 were similar.

table

Example  2 - humZ270  Competitive CD94 / NKG2A It is an antagonist.

HumZ270 was conjugated to CD94 / NKG2A-overexpressing Ba / F3 cells (Ba / F3-CD94), as described in U.S. Patent No. 8,206,709 to test whether humZ270 prevents ligand (ie, HLA-E) binding to CD94 / NKG2A / NKG2A) against the binding of HLA-E tetramers. HumZ270VL1 / VH1 having the heavy and light chains of each of SEQ ID NOS: 3 and 7 was used in this example, and for all of the other examples below, unless otherwise indicated, this antibody was also used as humZ270. For this, Ba / F3-CD94 / NKG2A was incubated with various concentrations of humZ270, or 2) with HLA-E tetramer (4.7 ㎍ / ml) . All incubations were performed on ice-cold, tissue-culture medium containing 2% FCS. Next, cells were incubated with APC-conjugated secondary antibodies specific for mouse Ab and analyzed by flow cytometry using BD Biosciences FACSarray.

humZ270 efficiently binds to Ba / F3-CD94 / NKG2A cells in a concentration dependent manner (lozenge). However, when the cells were pre-incubated with HLA-E tetramer, binding of humZ270 to Ba / F3-CD94 / NKG2A cells was prevented. Thus, HumZ270 and HLA-E bind to overlapping epitopes on CD94 / NKG2A. Thus, the CD94 / NKG2A-inhibiting effect of humZ270 in the NK-cytotoxicity assay may be the result of preventing the ability of HLA-E to induce a negative signal to cytotoxic lymphocytes through CD94 / NKG2A. Thus, humZ270 can be considered a competitive CD94 / NKG2A antagonist.

Example  3 - NKG2A  Expression-based rma - rae1  The tumor bearing C57 / bl6  Distribution of NK and T cell subset in mice

To further investigate the expression of NKG2A in the tumor environment, the distribution of NKG2A was studied in a subset of NK and T cells in mice. Lymphocytes were taken from the spleen, from tumor drain lymph nodes, and from solid tumors.

C57 / BL6 mice were transplanted into RMA-Rae clone 6 (2 million cells) (subcutaneous). These tumor cells express the CD94 / NKG2A ligand, Qa-1. Mice were sacrificed at day 12, with an average tumor volume of 723 ㎣, SD of 161 ㎣, and n = 4. After preparation of the cell suspension from spleen, LN and tumor, cells were stained as follows: CD3e PerCP Cy5.5, NKP46 Alexa 647, NKG2A / C / E FITC, CD8 Pacific Blue.

The results shown in Tables 1 to 3 reveal that NKG2A-expressing NK and CD8 + T lymphocytes are observed in a significant percentage in the tumor environment, while NKG2A + CD8 + T cells are present in high percentage in the tumor environment, This was not the case in the tumor drain lymph node. In the NK cell subset, approximately half of the cells in both draining lymph nodes and spleen were NKG2A-positive and half were NKG2A-negative. In the T cell subset, most cells were NKG2A-negative (only 1.1% in the lymph nodes and 4.7% in the spleen is NKG2A ⁺ ). In the CD8 T cell subset, most cells were again NKG2A-negative (only 1.6% in the lymph nodes and 3.9% in the spleen are NKG2A ⁺ ). However, while most of the CD8 T cells outside the tumor do not have NKG2A expression, a subset of tumor invading CD8 T cells had an average of 26.3% of NKG2A + positive cells. There was little difference in NKG2A expression observed between TIL and spleen or lymph node cells in the CD8 ^- T cell subset, since only 5.1% of CD8 ^- T cells expressed NKG2A in tumors.

Table 1: Spleen

Table 2: Tumor drain lymph node

Table 3: Tumor invading lymphocytes

Example  4 - In vitro  Receptor saturation: In whole blood NKG2A + NK  Binding of the humanized anti-NKG2A antibody IPH2201 to the cell

The receptor occupancy of anti-NKG2A was evaluated in vitro in human blood to provide a prediction of the pharmacokinetics of anti-NKG2A in human patients. These studies aimed to estimate the cell affinity of HumZ270 whole blood, and the absolute number of NKG2A receptors per ㎕ of whole blood and also per cell in humans in vitro. The affinity and total number of receptors can affect both the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs in humans. These data will be used as input to the PK / PD model to be used to design the initial human capacity test. Human whole blood was collected from each donor and processed immediately. Titration of humZ270 was performed on whole blood from 8 donors (mAb was incubated for 30 minutes at room temperature (RT)). Fifteen mAb concentrations were tested to cover the full range of binding from 0 to 100% of the maximal binding from 90 ug / ml to 0.000019 / / ml (1/3 step dilution, 15 points and 0). Samples were treated to remove red blood cells, the cells were fixed, and obtained from a cell calculator. The calibration beads were used to convert the MFI results to Molecular of Equivalent Soluble Fluorochrome (MESF). Gating was performed on CD45 + lymphocytes expressing NKG2A.

The three tubes were dedicated to absolute counting of a subset of lymphocytes using flow coefficient beads added into the blood. First, titration was performed using humZ270, where bound humZ270 was detected using PE-coupled anti-hIgG4 secondary antibody. In addition, to evaluate the total number of receptors for saturating concentrations of humZ270 and to test the effect of coupling to PE on the pharmacological properties of humZ270, titration with PE-coupled humZ270 was performed and the combined were compared with those titrated with humZ270.

Analysis focused on the NKG2A + cell subset, NK cells and T cells. None of these subsets of lymphocytes express NKG2A, thus studying the NKG2A-subset for each population of interest and comparing it to the NKG2A + subset. The cell population is defined as:

- lymphocytes: defined on the CD45 / SSC plot according to their size and size and CD45 expression.

- human NK lymphocytes: CD3-CD56 + cells in lymphocytes

- CD8 T cells: CD3 + CD8 + cells in lymphocytes

- NKG2A + lymphocytes: NKG2A + cells in lymphocytes

The total mean fluorescence intensity (MFI) was recorded for each population. Using the calibration beads, the fluorescence in the PE channel was expressed by MESF. The primary population of interest in these studies is NKG2A + lymphocytes, which mainly contain NK cells.

The results showed that the human anti-NKG2A mAb (humZ270) binds to a subset of two peripheral blood lymphocytes: NK and CD8 + T cells.

The results are summarized in FIG. HumZ270, also referred to as IPH2201, is represented by the different concentrations (ng / ml) listed on the x-axis and the MESF signal for receptor binding is shown on the y-axis. The antibody had a binding affinity (EC50) of about 4 ng / ml for NKG2A + cells (about 4 ng / ml of K _D ). This K _D is consistent with the K _D values observed in the other assays, particularly the affinity for binding to PBMC and affinity for recombinant NKG2A in the Biacore assay. The K _D (EC100) for complete receptor occupancy was about 100 ng / ml.

Example  5 - Rheumatism  In human phase I clinical trials in arthritis Of IPH2201  Receptor saturation

humZ270, a human IgG4 antibody was produced in heavy chain with serine to proline mutation (S241P mutation) at residue 241 of the heavy chain corresponding to position 228 according to the EU-index, in order to maintain high affinity divalent binding in vivo Respectively. The safety profile of humZ270 (IPH2201) was investigated in a double-blind, placebo-controlled dose-elevation Phase I study of 92 patients with stable and controlled rheumatoid arthritis. In this three-arm phase I study, Z270 or placebo is administered subcutaneously (subcutaneously or subcutaneously) in a single dose up to 10 mg / kg intravenously or in a single dose up to 4 mg / kg subcutaneously 2 weeks apart). The doses to be tested intravenously as a single dose were 0.0002, 0.001 mg / kg, 0.005 mg / kg, 0.025 mg / kg, 0.1 mg / kg, 0.4 mg / kg, 1.1 mg / kg, 3.5 mg / kg and 10 mg / Respectively. All patients were followed for at least 12 weeks. The safety profile was very favorable. MTD did not reach. There were no SUSAR, drug-related serious adverse events, infusion-related reactions, and immune-related adverse events. Headache and nasopharyngiomas were the two most common adverse events reported.

Receptor saturation was assessed using a conventional sandwich ELISA format and the human Fc human NKG2A fusion protein was used to capture the humZ270 antibody. After incubation with serum samples containing humZ270 antibody, the bound antibody (analyte in this study) is detected using biotinylated mouse anti-human IgG4. HRP-labeled avidin is added to tag solid-bound bound biotinylated anti-human IgG4 and a colorimetric HRP substrate (TMB) is used for endpoint detection. The optimal dose and frequency of administration for the anti-NKG2A (IPH2201) dose level to be used in clinical trials to provide substantially complete receptor saturation was determined using a model based on clinical data from IPH2201 Phase I in RA patients, a pharmacokinetic software package (WinNonLin 6.3 0.0 > Parsight < / RTI > Corporation). In clinical trials with IPH2201, the frequency of administration depends on the steady-state plasma concentration required for saturation, and the clearance and distribution volume of IPH2201.

IPH2201 Phase I clinical trials, preliminary PK data for intravenous injection and NCA analysis can allow the inventors to estimate the PK parameters for the PK model. Nominal time was used. A standard two-compartment model was selected using nonlinear target mediated drug placement and primary elimination modeled by Michaelis Lieberstein mechanics. A dose-dependent clearance was observed in the NCA analysis and applied to this model, and the clearance decreased as the dose increased below 0.4 mg / kg and remained constant for higher doses. This preliminary PK model is consistent with a PK characteristic for which dose dependence is known for IgGs that reflect target receptors (low dose) and FcRn receptor (high dose) saturation (Brambell).

Population PD modeling was performed based on preliminary NKG2A occupancy data available from ongoing Phase I clinical trials.

Using the S-shaped Emax model, the plasma concentration-effect relationship was fitted. The model is characterized by an EC ₅₀ , defined as the serum drug concentration, which achieves 50% of E _max , i. E., 50% of NKG2A occupation here.

However, when plotting PD data against PK data, it has been observed that as time after administration increases, the maximum NKG2A occupancy (Emax) obtained for the highest IPH2201 concentration decreases in a time dependent, . Similarly, EC50 appeared to increase linearly with time. To model this observation in the NKGA2 occupancy profile, NKG2A occupancy data was generated, including a decrease in Emax over time and an increase in EC50.

NKG2A _{occupied% = (E max - (E} max Fall × TSLD)) × C γ / ((EC 50 + EC 50 inc × TSLD) γ + C γ)

Where TSLD is the time since the last administration; E _max is 100%; E _max is the rate of decrease of the Emax F _all with time; EC ₅₀ inc is the growth rate of EC ₅₀ .

The results of the modeling are shown in Figure 2, and substantially complete (at least 90%) receptor saturation of NKG2A + cells can be achieved by administering 0.03 mg / kg every two weeks. A dose of 0.1 mg / kg may be administered every 4 weeks to maintain substantially complete saturation of NKG2A. When saturation is desired in extravascular tissues (e.g., solid tumors), approximately 10-fold higher doses are deemed to be necessary, which implies a dose of about 0.4 mg / kg every 2 weeks and 1 mg / kg every 4 weeks do.

The results for saturation of the NKG2A receptor from these human clinical trials were consistent with the concentration of anti-NSG2A (EC100) required for complete receptor occupancy determined in vitro (see Example 4).

Example  6 - IPH2201  Used NKG2A  CD107 response to blocking

In-vitro experiments with effectors and target cells were performed to assess the death mediated by cytokine-activated purified NK cells (effector cells) in 12 human subjects. In three donors, evaluation of mobilization and occupancy of CD107 was performed in parallel. The target cell was self-SEB blast (balst). Such killing is believed to be mediated by NK cells and the killing may be specifically increased by the anti-NKG2A antibody as the anti-NKG2A antibody is expected to correspond to an inhibitory signal from HLA-E. Concentration-range responses using the antibody humZ270 were performed in a 4-hour cytotoxicity assay. CD107 mobilization assays based on the results of the generated experiments and using purified NK cells were selected. First, FACS-based CD107 assays enable the study of the cytotoxic response of NK cells specifically expressing NKG2A and comparing it to NK cells that do not express NKG2A. By alternative protocols, for example, chromium release assays, it will not be possible to identify effects depending on the NKG2A expression in question in donors with low percentages of NKG2A + cells.

Frozen PBMCs were incubated with 200 IU / ml recombinant human IL-2 (Proleukin) and 100 ng / ml SEB (Staphylococcus) in complete medium (RPMI, 10% FCS, penicillin / streptomycin) Toxin B, < RTI ID = 0.0 > Sigma). ≪ / RTI > CD4 T cells were then negative selected by using "Stemsep negative selection human CD4 T cell enrichment kit" (Item # 14052A). Purity was assessed by FACS and HLA-E expression was measured by PE-conjugated anti-HLA-E clone 3D12 (E-Biosciences). T CD4 SEB blast was considered to be an excellent model for generating autologous cells mimicking autoreactive T CD4 + cells.

Purified NK cells were prepared from frozen PBMC: human NK was purified using the Stem Sep system and cultured overnight in complete medium supplemented with 10 IU / ml recombinant IL-2. PBMC were prepared from the collected blood. Blood was quantified for transfusion, so the donor is anonymous and presumably healthy. PBMC were prepared, aliquoted and stored in a nitrogen tank prior to the study.

The calculation of EC ₅₀ for the KD and CD107 mobilization for receptor saturation was carried out on the graph pad prism (Prism GraphPad) software using a non-linear regression fit with a four parameters. To estimate the affinity constant Kd from titration curves of anti-NKG2A (IPH2201) binding to purified NK cells, values for 30 and 90 占 퐂 / ml were excluded because some artifacts at these concentrations were excluded, .

The results are summarized in FIG. Surprisingly, the concentration of anti-NSG2A required for efficacy (blocking) is 100 times higher than the concentration that provides complete receptor saturation. In the CD107 mobilization assay, the EC50 concentration (the amount providing 50% of the maximal value) was determined to be about 400 ng / ml and the EC100 was about 10,000 ng / ml (10 ug / ml). In contrast, as shown in Example 4, the EC50 concentration for receptor saturation was about 4 ng / ml, the EC100 was about 100 ng / ml, the concentration also provided by data from Phase I clinical studies (see Example 5) )to be.

In the three donors viewing in parallel to CD107 mobilization and occupied, individual EC ₅₀ values for CD107 was higher about 100 times that of an individual Kd for the receptor saturation. The same ratio was observed when evaluating all data (median Kd 0.005 ug / ml for receptor saturation, n = 5, median EC50 for CD107 mobilization 0.40 ug / ml, n = 9). This suggests that the donors generally have similar receptor affinities for IPH2201 and that the relationship between receptor saturation and biological activity is similar.

Example  7 - NKG2A  For the CD107 response to blocking HLA Effect of -E expression level

A 100-fold difference between Kd obtained for receptor saturation and EC for CD107 mobilization can be potentially explained by membrane-bound HLA-E on the target cell in the CD107 assay, and membrane-bound HLA-E on the target cell More IPH2201 is required to reach the functional saturation of the receptor than to the receptor saturation experiment, which binds to NKG2A on NK cells with high binding force and does not involve HLA-E.

To investigate this possibility, the ECs observed for biological effects in the self-SEB blast of Example 6 were compared to ECs of cells with different levels of HLA-E expression. Human K562 cells transfected with HLA-E were selected as HLA-E highly expressing cells. FACS-based CD107 assays were performed using purified NK cells as described in Example 6.

In each experiment, autologous CD4 + SEB blasts and selected K562-HLA-E clones were tested for HLA-E expression using PE-coupled anti-HLA-E mAb, 3D12. HLA-E expression on K562HLA-E transfectants was at least 20-fold higher than constitutive HLA-E expression observed on autologous SEB blasts. The purity of the SEB blast was 96% ± 0.818 (SD, n = 12, range 94.6% to 97.1%). Expression of T CD4 SEB blast and HLA-E on K562-HLA-E was similar among all donors.

By providing this observation of a 100-fold difference between Kd obtained for receptor saturation and EC for CD107 mobilization, the biological effect on SEB blast compared to the EC50 on the K562-HLA-E transfectant (median 4.1 [mu] g / (Median 0.40 < RTI ID = 0.0 > g / ml) < / RTI > can be explained by at least 20-fold difference in HLA-E expression on the cell surface. In this case, higher quantities of IPH2201 are required to compete with HLA-E when compared to autologous SEB blast, since HLA-E is expressed at higher density on K562-HLA-E transfectants.

Example  8 - Repeated Of IPH2201  Prediction of pharmacokinetic models for injections

Based on the EC for CD107 mobilization observed for autologous (SEB) cells in Example 7, and incorporating the blood concentration results from the Step I test described in Example 5, modeling is performed to determine the efficacy of the clinical The optimal dose frequency for the anti-NSG2A (IPH2201) dose level used in the study was determined.

The optimal dose and frequency of administration for the anti-NKG2A (IPH2201) dose level to be used in clinical trials was assessed using a PK / PD model developed using a pharmacokinetic software package (WinNonLin 6.3.0.395, Phantom Corporation) And predicted using models based on clinical data from Phase I of IPH2201. In clinical trials with IPH2201, the frequency of administration depends on the steady-state plasma concentration needed and the volume of clearance and distribution of IPH2201.

IPH2201 Phase I clinical trials, preliminary PK data for intravenous injection and NCA analysis can allow the inventors to estimate the PK parameters for the PK model. Nominal time was used. A standard two-compartment model was selected using nonlinear target mediated drug placement and primary elimination modeled by Michaelis Lieberstein mechanics. A dose-dependent clearance was observed in the NCA assay and applied to this model, and the clearance decreased as the dose increased below 0.4 mg / kg and remained constant for higher doses. This preliminary PK model is consistent with a PK characteristic for which dose dependence is known for IgGs that reflect target receptors (low dose) and FcRn receptor (high dose) saturation (Brambell).

Based on the preliminary PK and PD data from the clinical phase I test, the following PK and PD parameters fit the observed data optimally:

Figure 3b shows that different blood concentrations are achieved at different doses administered intravenously every two weeks. The dose levels were 0.01, 0.02, 0.03, 0.04, 0.2, 0.4 mg / kg, 2 mg / kg, 4 mg / kg and 10 mg / kg (body weight). In Fig. 3B, the line corresponds to IPH2201 with increasing capacity from the lower side to the upper side, where the lowest capacity corresponds to the lowest line on the lower side of the chart and the highest capacity corresponds to the upper side of the chart. A dose of 0.01 mg / kg provided an initial (peak) blood concentration of about 0.2 [mu] g / ml. A dose of 0.04 mg / kg provided an initial (peak) blood concentration of greater than EC ₅₀ for efficacy of about 1 μg / ml, ie 400 ng / ml, but less than EC ₅₀ after two weeks. A dose of 0.4 mg / kg provided an initial (peak) blood concentration of approximately 10 μg / ml, ie approximately EC ₁₀₀ for efficacy in the circulation, and a sustained blood concentration of greater than 3 μg / ml up to 2 weeks. A dose of 4 mg / kg provided an initial (peak) blood concentration of about 100 μg / ml, ie, about EC ₁₀₀ for efficacy in tissues, and a sustained blood level of more than 30 μg / ml up to two weeks. However, when a dose of 4 mg / kg is administered repeatedly, the fourth dose provides a sustained serum concentration of approximately 100 [mu] g / ml. A dose of 10 mg / kg provided a sustained serum concentration of approximately 100 [mu] g / ml.

Figure 3c shows different blood concentrations achieved with different doses administered intravenously every two weeks when loading and retention capacity is used. Following the loading dose of 10 mg / kg (body weight), the maintenance dose of 4 mg / kg (body weight) (top line in Fig. 3c) provides an initial and sustained blood concentration of approximately 100 ug / do. For comparison, a fixed dose of 4 mg / kg is indicated by the lower line in Fig. 3c, indicating a continuous (or minimal) blood concentration of at least 30 [mu] g / ml between the initial injection and the subsequent injection of the second week, (Or minimal or residual) blood concentration of at least 50 μg / ml and at least 60-70 μg / ml at two weeks after the third injection at two weeks after two injections .

Example  9 - NK  Anti- NKG2A  Effect of dose-response

The immunotherapeutic approach to HNSCC is particularly complicated by the complete immunosuppression induced by HNSCC, which potentially reduces the efficiency of immunostimulatory efforts (see, for example, Duray et al. (2010) Clin. Dev Immunol. 2010: 1-15). The purpose of this experiment was to investigate whether anti-NKG2A antibodies targeting NKG2A could remove HNSCC cells.

The effect of the dose-response of anti-NKG2A on NK cell activation was determined by analysis of CD107 and CD137 expression. CD107 mobilization at the fourth time is a marker of release of soluble granules by NK cells (Alter et al., (2004) J Immunol Methods 294 (1-2): 15-22). The increase in CD137 expression at 24 hours correlates with the activation of several lymphocytes including NK cells (Kohrt et al. (2011) Blood 117 (8): 2423-2432). Analysis of CD107 and CD137 expression was performed on NK cells expressing or not expressing NKG2A (NKG2A + NK cells or NKG2A-NK cells, respectively). Since the antibody targets NKG2A, the effect of the antibody is expected to be observed only in NKG2A + NK cells, so that NKG2A-NK cells can be regarded as an internal control in the experiment.

The effector cells used were freshly isolated PBMC from healthy volunteers and the target cells were clones of the HNSCC cell line or the K562 cell line transfected with HLA-E. Cells were counted and passaged every 2 days in complete medium. Survival was measured and had to exceed 90%. They were kept in culture until passage 12. One day prior to the experiment, cells were counted and adjusted to 100,000 cells / well. Survival was measured and had to exceed 90%.

K562 cell lines were K562 clone E6 (HLA-E positive, CD32 low) and K562 clone F7 (HLA-E negative / low, CD32 low). Human head and neck cancer cell lines were screened by flow cytometry for expression of HLA-E (see Table C below). Three cell lines were selected for functional testing: FaDu (ATCC # HTB-43), H-N (DSMZ # ACC 417) and CAL-27 (DSMZ # ACC 446).

Table C

The effector cells used were freshly isolated PBMC from healthy volunteers. Target cells are HNSCC cell lines (FaDu, HN and CAL-27), and a clone (clone E6 = HLA-E ^+, clones F7 = HLA-E ^-) of the K562 cell line was transfected with HLA-E was of 2.5 / 1 E: T ratio was used. The readout was CD107 at the 4th hour versus CD137 at the 24th hour. Seven donors (FaDu and HN) and two donors (CAL-27) were tested. The heavy chain amino acid sequence is shown in SEQ ID NO: 3 and the anti-NSK2A antibody humZ270 VH1 with the light chain amino acid sequence shown in SEQ ID NO: 7 was used at a final concentration of 10 g / ml.

Figures 4a and 4b show CD107 (top) on NKG2A-NK (left) or NKG2A + NK cells (right) in the presence of control or labeled target cell lines and in the presence or absence of anti- And CD137 (lower) FACS readings. Cell lines were sorted from left to right according to the level of HLA-E surface expression. Each point represents PBMC from healthy volunteers. Figure 4b shows the HNSCC cell line and demonstrates that anti-NSG2A can restore the dissolution of K562 transfected with HNSCC or HLA-E with endogenous HLA-E expression. This effect is only observed on NKG2A-positive NK cells and is dependent on the level of expression of HLA-E. Indeed, the anti-NKG2A effect is observed in cell lines with moderate to high HLA-E expression levels.

Anti-NKG2A can induce NK-mediated lysis of HLA-E expressing cell lines by blocking the interaction of inhibitory receptors NKG2A and HLA-E. This effect is observed on K562 cell lines transfected with HLA-E, but it is more important to be observed on HNSCC cell lines, such as FaDu and CAL-27 cell lines, with endogenous HLA-E expression. The extent of the anti-NKG2A effect depends on the level of HLA-E expression on the cell surface of the target cell.

Example  10 - Optimal dose of anti- NKG2A and  Lane capacity Cetuximab  Combined effect

The epithelial growth factor receptor (EGFR) (also ErbB-1 (HER1 in humans) is a transmembrane glycoprotein that is ubiquitously expressed in receptor tyrosine kinases with ErbB / HER. High expression of EGFR occurs in most epithelial malignancies including HNSCC and is associated with poor prognosis. Activation of EGFR through natural ligands regulates cell proliferation, invasion, activation of angiogenesis and metastasis, leading to initiation of intracellular signaling pathways leading to tumor growth.

The anti-EGFR monoclonal antibody, cetuximab, is believed to act by blocking the carcinogenic signaling of the EGF receptor pathway and by inducing Fcγ receptor-mediated antibody-dependent cellular cytotoxicity (ADCC). However, in HNSCC, ADCC can be affected by extreme immunosuppression induced. At the same time, the blockade of carcinogenic signaling of the EGF receptor pathway is mediated by the major histocompatibility complex (MHC) class I-related antigen, MICA / B and ULBP protein, recognized by the activation receptor NKG2D on NK cells and a subset of T- Lt; RTI ID = 0.0 > transcriptional < / RTI > In particular, expression of these stress-associated antigens, which are natural ligands of NKG2D, by tumor cells is reduced by clinical EGFR inhibitors, thereby potentially reducing the visibility of tumor cells to NK and T cells (Vantourout et al., Sci. Transl. Med. 6: 231 la49 (2014)].

These experiments were designed to investigate the effect of EGFR inhibiting antibodies on the ability of anti-NKG2A antibodies to activate NK cells in HNSCC. The dose-response effect of cetuximab on NK cell activation was used as effector cells, freshly isolated PBMC from healthy volunteers, and HNSCC cell lines (FaDu and HN) as target cells with an E: T ratio of 2.5 / 1 ≪ / RTI > CD107 and CD137 expression. The readings were CD107 at 4 hours versus CD137 at 24 hours using 3 donors (CD107 readings in 4 hours) and 4 donors (CD137 readings within 24 hours). Cetuximab was tested in a 1/10 step dilution starting at 10 μg / ml in the dose response. In one healthy volunteer, NK cells were subdivided into NKG2A + and NKG2A-subset. Lane dose of cetuximab was selected for further testing to investigate the effect of EGFR inhibiting antibodies on the ability of anti-NSK2A antibody to activate NK cells in HNSCC. A dose of 0.001 [mu] g / ml (1 ng / ml) is the starting point of the cetuximab effect observed with both CD107 and CD137 readings. A dose of 0.01 [mu] g / ml (10 ng / ml) is approximately an EC50 of cetuximab effect.

The combined effect of anti-NSG2A and lane dose of EGFR inhibitor was used as effector cells, freshly isolated PBMC from healthy volunteers and HNSCC cell lines (FaDu, HN and CAL-27) as target cells, K562 cell line transfected with HLA-E clones (clone E6 = HLA-E ^+, clones F7 = HLA-E ^-) of the E 2.5 / 1 was evaluated by analysis of CD107 and CD137 expression using a ratio T. The readings were CD107 at 4 hours and CD137 at 24 hours using 7 donors (FaDu and HN)) and 2 donors (CAL-27).

The heavy chain amino acid sequence is shown in SEQ ID NO: 3 and the light chain amino acid sequence shown in SEQ ID NO: 7 is used at a final concentration of 10 μg / ml, corresponding to a complete functional activity, and the EGFR inhibitor Cetux Simps were used in two lane doses of 0.001 [mu] g / ml or 0.01 [mu] g / ml.

The full active concentration of anti-NSG2A promoted ADCC for the HNSCC cell line induced by the later dose of cetuximab. Cetuximab-dependent ADCC was not observed in the K562 transfected cell line because the K562 transfected cell line does not express EGF-R. The anti-NKG2A effect is observed only in NKG2A-positive NK cells and is dependent on the level of HLA-E expression. Indeed, the anti-NKG2A effect is observed in cell lines with HLA-E with moderate to high expression levels (FaDu, CAL-27 and K562-HLA-E clone E6). Figure 5 is a representative example of FaDu cells. In Fig. 5, it can be observed that a fully active dose of anti-NSG2A enhances ADCC for HNSCC cell lines induced by corteximab (ctx) of the lane dose.

Example  11 - Increasing dose of anti- NKG2A and  Of increasing capacity Cetuximab  Combined effect

The effect of increasing doses of EGFR inhibiting antibody and increasing dose of anti-NSG2A antibody was evaluated for the ability to activate NK cells against HNSCC target cells. The experiment was to evaluate whether anti-NSG2A therapy could still enhance ADCC when cetuximab was used in saturating doses and whether the anti-NSG2A effect was dose-dependent.

Briefly, the effector cells used were freshly isolated PBMC from healthy volunteers and the target cells were clones of HNSCC cell line (FaDu, HN and CAL-27), and K562 cell line transfected with HLA-E (clone E6 = HLA- E ^+, clones F7 = HLA-E ^- was), E: T ratio was 2.5 / 1. The readings were CD107 at 4 hours and CD137 at 24 hours using 2 donors. Wherein the heavy chain amino acid sequence is shown in SEQ ID NO: 3 and the light chain amino acid sequence is set forth in SEQ ID NO: 7 in two lane doses of 0.1 and 1 [mu] g / ml and 10 [mu] g / Respectively. Cetuximab was used in these experimental settings in two lane doses of 0.001 [mu] g / ml and 0.01 [mu] g / ml (approximately EC50) and at a saturation dose of 0.1 [mu] g / ml.

The results are shown in Figures 6A and 6B. Figure 6a shows CD107 readings for a control with and without a K562-HLA-E transfectant. Each healthy volunteer is represented by a different symbol: square or circle. The white symbols with crosses correspond to conditions in which the anti-NKG2A was replaced with a 10 [mu] g / ml hIgG4 isotype control co-incubated with 0.1 [mu] g / ml cetuximab. The effect of anti-NKG2A antibody is dose-dependent and can be observed to be dependent on HLA-E expression level, and in clone E6 expressing HLA-E at high levels, in a saturated dose of 0.1 μg / ml cetuximab Still observed. In clone F7 (low HLA-E expression levels), the effect of anti-NSG2A was limited and high dose (e.g., full active dose) of anti-NSG2A did not further increase the effect.

Figure 6b shows CD107 readings for HNSCC cell lines. Each healthy volunteer is represented by a different symbol: square or circle. White symbols with crossed lines correspond to conditions in which anti-NKG2A was replaced by a 10 [mu] g / ml hIgG4 isotype control co-incubated with 0.1 [mu] g / ml cetuximab. The effect of anti-NKG2A antibody is dose-dependent and can be observed to be dependent on HLA-E expression level and can be observed in FaDu or CAL-E expressing HLA-E at a higher level / strongly stained for HLA- Lt; RTI ID = 0.0 > g / ml < / RTI > of cetuximab.

The effect of anti-NKG2A antibody is dose-dependent and is dependent on HLA-E expression levels and is still observed with a saturation dose of 0.1 μg / ml of cetuximab.

Example  12 - Human clinical trial for the treatment of cancer using repeated injections of humanized Z270 as a single agent

The primary objective of the trial is to evaluate the antitumor activity of pre-operative IPH2201 (humanized Z270, including the S241P mutation) in patients with operable squamous cell carcinoma of the mouth. The secondary objective is to evaluate the pharmacodynamic characteristics of IPH2201, including safety, pharmacokinetic characteristics, immunogenicity and tumor biomarkers.

Test design:

The test is a single-organ, open label single-arm phase Ib-II study involving the introduction. Previously untreated patients with measurable clinically intermediate or high-risk oral or IVa oral squamous cell carcinoma were treated intravenously (iv) every 2 weeks (q2w) for 4 doses by intravenous (iv) Will be treated with a single agonist IPH2201. The first 6 patients will receive IPH2201 at a dose of 4 ㎎ / ㎏ × 2 times every 2 weeks. A minimum interval of one week between IPH2201 administration of the first three patients treated with 4 mg / kg will be observed. Subsequent patients will be treated × 4 times every 2 weeks with a dose of 10 ㎎ / ㎏, and the dose escalation will be allowed by the safety committee after a minimum of 4 weeks following the first dose in the last patient treated with 4 ㎎ / ㎏ . Following the last administration of IPH2201, standard topical treatment with surgery will be followed by adjuvant therapy (radiation therapy (RT) or radiation chemotherapy (RCT)) according to the histopathological risk factors. In the case of tumor progression, topical treatment will be initiated immediately.

The antitumor activity will be assessed clinically and radiologically before the third administration of IPH2201 and two weeks after the last administration of IPH2201 prior to surgery. Tumor measurements will be obtained in target lesions according to RECIST 1.1 criteria. The same imaging technique will be used at baseline and for preoperative evaluation, for evaluation of primary endpoints, and / or for monitoring possible recurrence after surgery. Assessment by appropriate imaging techniques (computed tomography (CT) scan and / or magnetic resonance imaging (MRI)) will be performed in all patients and in photographs of accessible tumor lesions at the discretion of the investigator.

Fresh tumor samples will be obtained by biopsy at baseline, and resected specimens will be collected at the time of surgery. Pharmacology (PK / PD) and biomarker research will be conducted before and after surgery.

The patient will be followed up to one year after the first administration cycle. After the last study visit, we will document recurrence and survival after the study according to local practice for an additional two years.

All references, including publications, patent applications, and patents, cited herein, are hereby incorporated by reference in their entirety to the same extent as if each appeared to be incorporated herein by reference in their entirety and each reference is individually and specifically incorporated by reference, (To the maximum extent permitted by law) are hereby described, whether or not the inclusion of a particular document in a given place is provided separately and selectively.

The use of the singular terms and similar terms in the description of the present invention should be construed to cover both the singular and the plural unless otherwise indicated herein or otherwise clearly contradicted by context.

Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all precise exemplary values provided for a particular factor or measurement are also appropriately represented as " about " ≪ / RTI > can be considered to be provided). When "about" is used in connection with a number, it may be specified to include a value corresponding to +/- 10% of a particular number.

The description herein of any aspect or embodiment of the invention that uses terms such as "comprising," "having, " or" containing ", when used herein, Is intended to provide support for similar aspects or implementations of the present invention, unless otherwise indicated, to " comprise, "" comprise," Should be understood as describing a composition consisting of such elements, unless otherwise stated or clearly contradicted by context).

The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the invention and is not intended to limit the scope of the present invention, It is not the case. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the invention.

                         SEQUENCE LISTING <110> INNATE PHARMA   <120> TREATMENT REGIMENS USING ANTI-NKG2A ANTIBODIES <130> IP20173126 / FR <150> US 62 / 050,948 <151> 2014-09-16 &Lt; 150 > US 62 / 067,642 <151> 2014-10-23 <150> US 62 / 083,929 <151> 2014-11-25 &Lt; 150 > US 62 / 093,141 <151> 2014-12-17 <150> US 62 / 093,124 <151> 2014-12-17 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 233 <212> PRT <213> Homo sapiens <400> 1 Met Asp Gln Gly Val Ile Tyr Ser Asp Leu Asn Leu Pro Pro Asn 1 5 10 15 Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Leu             20 25 30 Ala Thr Glu Glu Glu Ile Thr Tyr Ala Glu Leu Asn Leu Gln Lys Ala         35 40 45 Ser Gln Asp Phe Gln Gly Asn Asp Lys Thr Tyr His Cys Lys Asp Leu     50 55 60 Pro Ser Ala Pro Glu Lys Leu Ile Val Gly Ile Leu Gly Ile Ile Cys 65 70 75 80 Leu Ile Leu Met Ala Ser Val Val Thr Ile Val Valle Pro Ser Thr                 85 90 95 Leu Ile Gln Arg His His Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys             100 105 110 Ala Arg His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn         115 120 125 Ser Cys Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu     130 135 140 Leu Ala Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp Asn Glu 145 150 155 160 Glu Glu Met Lys Phe Leu Ser Ile Ile Ser Pro Ser Ser Trp Ile Gly                 165 170 175 Val Phe Arg Asn Ser Ser His His Pro Trp Val Thr Met Asn Gly Leu             180 185 190 Ala Phe Lys His Glu Ile Lys Asp Ser Asp Asn Ala Glu Leu Asn Cys         195 200 205 Ala Val Leu Gln Val Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser     210 215 220 Ile Ile Tyr His Cys Lys His Lys Leu 225 230 <210> 2 <211> 452 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 2 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr             20 25 30 Trp Met Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Pro Ser Phe     50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe             100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser     130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys         195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Leu Gly Lys     450 <210> 3 <211> 452 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Leu     50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe             100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser     130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys         195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Leu Gly Lys     450 <210> 4 <211> 452 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 4 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr             20 25 30 Trp Met Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Pro Ser Phe     50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe             100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser     130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys         195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Leu Gly Lys     450 <210> 5 <211> 452 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 5 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Glu Lys Phe     50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Thr Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe             100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser     130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys         195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Leu Gly Lys     450 <210> 6 <211> 452 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe             100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser     130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys         195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Leu Gly Lys     450 <210> 7 <211> 214 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 7 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Arg                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 8 <211> 5 <212> PRT <213> mus musculus <400> 8 Ser Tyr Trp Met Asn 1 5 <210> 9 <211> 11 <212> PRT <213> Mus musculus <400> 9 Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr 1 5 10 <210> 10 <211> 17 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 10 Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly      <210> 11 <211> 17 <212> PRT <213> Artificial <220> <223> Chimeric mouse-human <400> 11 Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Ala Gln Lys Leu Gln 1 5 10 15 Gly      <210> 12 <211> 16 <212> PRT <213> Mus musculus <400> 12 Gly Gly Tyr Asp Phe Asp Val Gly Thr Leu Tyr Trp Phe Phe Asp Val 1 5 10 15 <210> 13 <211> 11 <212> PRT <213> Mus musculus <400> 13 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 14 <211> 7 <212> PRT <213> Mus musculus <400> 14 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 15 <211> 9 <212> PRT <213> Mus musculus <400> 15 Gln His His Tyr Gly Thr Pro Arg Thr 1 5

Claims (48)

An antibody that binds to NKG2A and neutralizes its inhibitory activity for use in the treatment of disease in an individual, said treatment comprising administering said antibody to said subject for at least one administration cycle, wherein said anti-NSG2A Wherein the antibody is administered at least twice in an effective amount to achieve and / or maintain a blood level of at least 10 [mu] g / ml of anti-NSG2A antibody between two consecutive doses of anti-NSG2A antibody. 2. The antibody of claim 1, wherein said anti-NSK2A antibody is administered at least twice in an effective amount to maintain a sustained blood level of the anti-NSK2A antibody at least 10 &lt; RTI ID = 0.0 &gt; The method of claim 1 or 2 wherein said antibody is administered to maintain a sustained serum concentration of anti-NSG2A antibody of at least 10 [mu] g / ml, alternatively from 10 to 100, 10 to 80 or 10 to 50 [ &Lt; / RTI &gt; 4. The method according to any one of claims 1 to 3, wherein said antibody is administered at the time of administration to achieve a peak blood concentration of the anti-NSG2A antibody of at least 40 [mu] g / ml, 50 [ Antibody. 5. The method of any one of claims 1 to 4 wherein said antibody is at least about 40, 50, or 100 &lt; RTI ID = 0.0 &gt; g / ml &lt; / RTI & Wherein said antibody is administered in an effective amount to provide a sustained (minimal) blood concentration of the anti-NKG2A antibody. 6. The method of any one of claims 1 to 5 wherein said antibody is administered to maintain a sustained (minimum) blood level of anti-NSG2A antibody of at least about 100 [mu] g / ml between two consecutive doses of anti- Antibody. 7. The method of any one of claims 1 to 6 wherein said antibody is administered during two consecutive doses of an anti-NKG2A antibody, optionally during the course of dosing, at a dose of at least 100 μg / ml, alternatively 100-200 μg / Wherein the antibody is administered to maintain a sustained serum concentration of the anti-NKG2A antibody. 8. The method according to any one of claims 1 to 7, wherein said antibody is administered in an effective amount to maintain a sustained serum concentration of anti-NSG2A antibody between at least 40 [mu] g / ml of two consecutive doses of anti-NSG2A antibody , Antibodies. 9. The antibody according to any one of claims 1 to 8, wherein said antibody is administered twice a month. 10. A method according to any one of claims 1 to 9, wherein said antibody is administered intravenously twice a month and comprises at least 10 [mu] g / ml of anti-NSK2A antibody between at least two consecutive doses of anti- Wherein the amount is 2 to 10 mg / kg, optionally 2 to 6 mg / kg, alternatively 2 to 8 mg / kg or alternatively 2 to 4 mg / kg (body weight). 11. The method according to any one of claims 1 to 10, wherein said antibody is administered intravenously twice a month and comprises at least 100 mu g / ml of anti-NSG2A antibody between at least two consecutive doses of anti- Kg, alternatively about 6 mg / kg, alternatively about 8 mg / kg, alternatively about 4 mg / kg, alternatively about 4 mg / kg, alternatively about 4 mg / Optionally about 10 mg / kg body weight. 12. The method of any one of claims 1-11, wherein said treatment is administered at least once at an initial effective dose to maintain a blood level of at least 100 [mu] g / ml until the next sequential administration of the anti-NSG2A antibody Followed by a period of time during which the antibody is administered at a frequency that is effective to maintain a sustained blood level of the anti-NSG2A antibody of at least 100 [mu] g / ml of the anti-NSG2A antibody between consecutive doses of the anti- &Lt; / RTI &gt; 13. The method of claim 12, wherein the antibody is administered intravenously and the loading period comprises administering the antibody once with a dose of 8-10 mg / kg, optionally about 10 mg / kg, Optionally 2 mg / kg, alternatively 2 mg / kg, alternatively 2 mg / kg, alternatively 2 mg / kg, alternatively 2 mg / kg, alternatively about 3 mg / kg, alternatively about 4 mg / At a dose of about 6 mg / kg (body weight), at least twice, at intervals of about 2 weeks. An antibody that binds to NKG2A and neutralizes its inhibitory activity for use in treating an individual, said treatment comprising administering said antibody to said subject for at least one administration cycle, wherein said anti-NKG2A antibody 4 to 10 mg / kg, alternatively 4 to 6 mg / kg, alternatively 4 to 8 mg / kg, alternatively about 4 mg / kg, alternatively about 6 mg / kg, alternatively about 8 mg / To a dose of about 10 mg / kg (body weight). An antibody that binds to NKG2A and neutralizes its inhibitory activity for use in treating an individual, said treatment comprising administering said antibody to said subject for at least one administration cycle,
a. A loading period during which the antibody is administered intravenously at least once with an initial dose of 8-10 mg / kg, optionally about 10 mg / kg, and
b. Optionally, about 2 mg / kg, optionally about 3 mg / kg, alternatively about 2 mg / kg, alternatively about 2 mg / kg, alternatively about 2 mg / Kg, or alternatively at least about 6 mg / kg body weight, at least twice every two weeks; optionally, wherein the first administration within the maintenance period is less than 2 weeks after the initial dose &Lt; / RTI &gt; 16. The method of claim 15, wherein the antibody is administered at a frequency that is effective to maintain a sustained serum concentration of anti-NSG2A antibody of at least 10 [mu] g / ml, alternatively at least 40 g / ml, 50 g / &Lt; / RTI &gt; 17. The antibody of any one of claims 1 to 16, wherein said administration cycle is 8 weeks or at least 8 weeks. 18. The antibody of any one of claims 1 to 17, wherein said administration cycle comprises at least 4 administrations of an anti-NSG2A antibody. 19. The antibody of any one of claims 1 to 18, wherein the subject has a cancer. 20. The antibody of claim 19, wherein the subject has a haematological tumor. 20. The antibody of any one of claims 1, 2, or 4 to 19, wherein the subject has a solid tumor. 22. The antibody of claim 21, wherein the subject has HNSCC. 23. The antibody of any one of claims 1 to 22, wherein said individual has a solid tumor and is treated with an anti-NSK2A antibody prior to surgery to remove the tumor. 24. The antibody of claim 23, wherein said individual is treated with a full dosage cycle of anti-NSG2A antibody prior to surgery to remove the tumor. 25. The antibody of any one of claims 1 to 24, wherein said antibody competes with HLA-E for binding to NKG2A. Claim 1 to claim 25, wherein according to any one of, wherein the antibody is an antibody having an EC ₅₀ of from 1 to 10 ng / ㎖ for binding to the NKG2A- expressing cells. 27. The antibody of any one of claims 1 to 26, wherein said antibody has a K _D of 10 ^-10 M to 10 ^-12 M for binding to soluble recombinant NKG2A polypeptide. 28. The antibody of any one of claims 1 to 27, wherein said antibody comprises a human IgG4 constant region. 29. The antibody of any one of claims 1 to 28, wherein the antibody comprises an Fc-engineered constant region comprising an amino acid modification that reduces binding to a human Fc? Receptor. 30. The antibody of any one of claims 1 to 29, wherein said antibody is an antibody fragment. 31. The antibody of claim 30, wherein the antibody fragment lacks an Fc domain. 32. The method of claim 30 or 31 wherein said antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, Fab'-SH fragment, F (ab) ² fragment, F (ab') ² fragment, Fv fragment, Camel Ig), a V _HH fragment, a single domain FV, and a single chain antibody fragment. 33. The antibody according to any one of claims 1 to 32, wherein the antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOS: 2 to 6 and a light chain sequence of SEQ ID NO: 7 for binding to human NKG2A &Lt; / RTI &gt; 34. The antibody of any one of claims 1 to 33, wherein the antibody comprises the CDR1, CDR2 and CDR3 domains of the heavy chain having the sequence set forth in SEQ ID NO: 2, and CDR1 of the light chain having the sequence of SEQ ID NO: 3 , CDR2 and CDR3 domains. 34. The antibody of any one of claims 1 to 34, wherein the antibody comprises a VH CDR of a heavy chain comprising the amino acid sequence of any one of SEQ ID NOS: 2 to 6 and a light chain comprising the amino acid sequence of SEQ ID NO: RTI ID = 0.0 &gt; CDR, &lt; / RTI &gt; 35. The antibody of any one of claims 1 to 35, wherein said anti-NSG2A antibody is administered as a pharmaceutically acceptable composition comprising a therapeutically effective amount of an anti-NSG2A antibody. 37. The antibody of claim 36, wherein the composition is free of any other pharmaceutically active agent. a) determining the HLA-E polypeptide status of malignant cells in a subject having cancer, and
b) determining a blood (serum) concentration of the anti-NSG2A antibody corresponding to at least an EC ₅₀ for an NKG2A + NK cell response, optionally an EC ₁₀₀ , in determining that the patient has an HLA-E polypeptide that is significantly expressed on the surface of malignant cells &Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;
An antibody that binds to NKG2A and neutralizes its inhibitory activity for use in the treatment or prevention of cancer in an individual. 39. The method of claim 38, wherein the step of determining the HLA-E polypeptide status in step (a) comprises determining the level of expression of a HLA-E nucleic acid or polypeptide of a malignant cell in a biological sample and comparing the level with a reference level &Lt; / RTI &gt; 41. The method of claim 38 or 39 wherein said antibody is administered in an amount effective to achieve an extracellular tissue concentration of an anti-NSK2A antibody corresponding to at least an EC ₅₀ , and optionally an EC ₁₀₀ , for an NKG2A + NK cell response. 41. The method of any one of claims 38 to 40, wherein the EC ₅₀ for NKG2A + NK cell response is about 4 [mu] g / ml. 41. A method according to any one of claims 38 to 40, wherein the EC ₁₀₀ for NKG2A + NK cell response is about 10 [mu] g / ml. 43. The method according to any one of claims 38 to 42, wherein said antibody competes with HLA-E for binding to NKG2A. For use in the treatment of diseases of the object, by binding to NKG2A as the antibodies that neutralize its inhibitory activity, the treatment NKG2A + NK least EC ₅₀ for the cell reaction, optionally for achieving blood levels equivalent to EC ₁₀₀ Comprising administering to said subject an effective amount of said antibody. 45. The method of claim 44, wherein the EC ₅₀ for the NKG2A + NK cell response is about 4 [mu] g / ml. 45. The method of claim 44, wherein the EC ₁₀₀ for the NKG2A + NK cell response is about 10 [mu] g / ml. 46. The method according to any one of claims 44 to 46, wherein said antibody competes with HLA-E for binding to NKG2A. 47. The method according to any one of claims 1 to 47, wherein the NKG2A + NK cell response is an NKG2A + NK cell response to an HLA-E expressing target cell.

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